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Novel (Phenylethynyl)pyrene–LNA Constructs for Fluorescence SNP Sensing in Polymorphic Nucleic Acid Targets
Author(s) -
Astakhova Irina Kira,
Samokhina Evgeniya,
Babu B. Ravindra,
Wengel Jesper
Publication year - 2012
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201200079
Subject(s) - oligonucleotide , nucleic acid , fluorescence , locked nucleic acid , dna , complementary dna , molecular beacon , microbiology and biotechnology , hybridization probe , rna , nucleic acid thermodynamics , biology , molecular probe , computational biology , chemistry , biochemistry , gene , physics , quantum mechanics
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.