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Nucleic Acids Research, 2002, Vol. 30, No. 21 e114
© 2002 Oxford University Press

Synthesis and evaluation of phosphorescent oligonucleotide probes for hybridisation assays

Paul J. O’Sullivan, Martina Burke, Aleksi E. Soini1 and Dmitri B. Papkovsky*

Biochemistry Department, University College Cork, Lee Maltings, Cork, Ireland and 1 Arctic Diagnostics Oy, BioCity A, 5th floor, Tykistökatu 6, 20521 Turku, Finland

*To whom correspondence should be addressed. Tel: +353 21 490 4257; Fax: +353 21 427 4034; Email: d.papkovsky{at}ucc.ie

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 (~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.


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