Triplex formation by pyrene-labelled probes for nucleic acid detection in fluorescence assays.
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 degrees C and in an increase in the fluorescence quantum yield (Phi(F)) from 0.061 to 0.179, while the probe alone was fluorescently silent (Phi(F)=0.001-0.004). To achieve higher triplex stability, five nucleobases in a 14-mer sequence were substituted with alpha-L-LNA monomers, which provided a triplex with a T(m) of 49.5 degrees C and a Phi(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 (Phi(F)=0.081) than for the triplex.
Van Daele
Ineke
I
Nucleic Acid Center, Department of Physics and Chemistry, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.
Bomholt
Niels
N
Filichev
Vyacheslav V
VV
Van Calenbergh
Serge
S
Pedersen
Erik B
EB
D020029:Base Pairing; D004247:DNA / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009696:Nucleic Acids / Q000032:analysis* / Q000737:chemistry; D009844:Oligosaccharides / Q000138:chemical synthesis / Q000737:chemistry; D011721:Pyrenes / Q000737:chemistry*; D011743:Pyrimidines / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods; D013696:Temperature; D014529:Uridine / Q000737:chemistry
D005456:Fluorescent Dyes; D009696:Nucleic Acids; D009844:Oligosaccharides; D011721:Pyrenes; D011743:Pyrimidines; C051802:triplex DNA; D004247:DNA; D014529:Uridine