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Triplex Formation by Pyrene‐Labelled Probes for Nucleic Acid Detection in Fluorescence Assays
Author(s) -
Van Daele Ineke,
Bomholt Niels,
Filichev Vyacheslav V.,
Van Calenbergh Serge,
Pedersen Erik B.
Publication year - 2008
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.200700533
Subject(s) - nucleic acid , pyrene , fluorescence , chemistry , molecular beacon , nucleic acid quantitation , biochemistry , nucleic acid detection , combinatorial chemistry , dna , oligonucleotide , organic chemistry , physics , quantum mechanics
Triplex‐forming homopyrimidine oligonucleotides containing insertions of a 2′–5′ uridine linkage featuring a pyrene moiety at the 3′‐position exhibit strong fluorescence enhancement upon binding to double‐stranded DNA through Hoogsteen base pairing. It is shown that perfect matching of the new modification to the base pair in the duplex is a prerequisite for strong fluorescence, thus offering the potential to detect single mutations in purine stretches of duplex DNA. The increase in the fluorescence signal was dependent on the thermal stability of the parallel triplex, so a reduction in the pH from 6.0 to 5.0 resulted in an increase in thermal stability from 25.0 to 55.0 °C and in an increase in the fluorescence quantum yield ( Φ F ) from 0.061 to 0.179, while the probe alone was fluorescently silent ( Φ F =0.001–0.004). To achieve higher triplex stability, five nucleobases in a 14‐mer sequence were substituted with α‐ L ‐LNA monomers, which provided a triplex with a T m of 49.5 °C and a Φ F of 0.158 at pH 6.0. Under similar conditions, a Watson–Crick‐type duplex formed with the latter probe showed lower fluorescence intensity ( Φ F =0.081) than for the triplex.