Synthesis and evaluation of phosphorescent oligonucleotide probes for hybridisation assays.
Monofunctional, p-isothiocyanatophenyl-derivatives of platinum (II)-coproporphyrin-I (PtCP-NCS) were evaluated as phosphorescent labelling reagents for synthetic oligonucleotides containing a 3'- or 5'-amino modification. Synthesis and purification conditions were optimised to generate high yields and purity of PtCP-labelled oligonucleotide probes. Phosphorescent properties of the PtCP label have been shown to be largely unaffected by conjugation to oligonucleotides of various length, GC composition and label attachment site. 5'-PtCP-labelled oligonucleotides were shown to work efficiently as primers in a standard PCR. A dedicated 532 nm laser-based time-resolved fluorescence plate reader enabled highly sensitive detection of PtCP-labelled oligonucleotides and PCR products, both in solution and in agarose gels, with limits of detection in the order of 0.3 pM. A model system employing two complementary oligonucleotides labelled with PtCP and QSY 7 dye (dark quencher) showed strong (approximately 20-fold) and specific proximity quenching of PtCP label upon hybridisation in solution. The potential applications of PtCP-labelled probes in hybridisation assays were discussed.
O'Sullivan
Paul J
PJ
Biochemistry Department, University College Cork, Lee Maltings, Cork, Ireland.
Burke
Martina
M
Soini
Aleksi E
AE
Papkovsky
Dmitri B
DB
D008163:Luminescent Measurements; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes / Q000138:chemical synthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D012996:Solutions
D015345:Oligonucleotide Probes; D012996:Solutions