Synthesis and evaluation of phosphorescent oligonucleotide probes for hybridisation assays

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Abstract
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.