title: Novel (phenylethynyl)pyrene-LNA constructs for fluorescence SNP sensing in polymorphic nucleic acid targets.
abstract: We describe fluorescent oligonucleotide probes labeled with novel (phenylethynyl)pyrene dyes attached to locked nucleic acids. Furthermore, we prove the utility of these probes for the effective detection of single-nucleotide polymorphisms in natural nucleic acids. High-affinity hybridization of the probes and excellent fluorescence responses to single-base mismatches in DNA/RNA targets are demonstrated in model dual-probe and doubly labeled probe formats. This stimulated us to develop two diagnostic systems for the homogeneous detection of a drug-resistance-causing mutation in HIV-1 protease cDNA and RNA gene fragments. Target sequences were obtained by analysis of 200 clinical samples from patients currently receiving anti-HIV/AIDS combination therapy at the Russian Federal AIDS Center. Using these fluorescent oligonucleotides, we were able to detect the target mutation despite all the challenges of the natural targets, that is, the presence of additional mutations, neighboring sequence variation, and low target concentration, which typically reduce binding and effectiveness of sensing by fluorescent oligonucleotides.
authors: Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, 5230 Odense M, Denmark. ias@sdu.dk
authors: Irina Kira
authors: IK
authors: Astakhova
authors: Evgeniya
authors: E
authors: Samokhina
authors: B Ravindra
authors: BR
authors: Babu
authors: Jesper
authors: J
authors: Wengel
mesh_terms: D020137:Base Pair Mismatch; D001483:Base Sequence; D005456:Fluorescent Dyes / Q000737:chemistry; D016333:HIV Protease / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000378:metabolism; D009841:Oligonucleotides / Q000737:chemistry*; D020641:Polymorphism, Single Nucleotide; D011721:Pyrenes / Q000737:chemistry*; D012313:RNA / Q000032:analysis; D013050:Spectrometry, Fluorescence
chemical_list: D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D009841:Oligonucleotides; D011721:Pyrenes; C477371:locked nucleic acid; D012313:RNA; C030984:pyrene; D016333:HIV Protease; C514803:p16 protease, Human immunodeficiency virus 1