An oligonucleotide probe incorporating the chromophore of green fluorescent protein is useful for the detection of HER-2 mRNA breast cancer marker.

Diagnosis and treatment of breast cancer can be greatly enhanced and personalized based on the quantitative detection of mRNA markers. Here, we targeted the development of a fluorescent oligonucleotide probe to detect specifically the HER-2 mRNA breast cancer marker. We have selected the chromophore of the Green Fluorescent Protein (GFP), 4-hydroxybenzylidene imidazolinone (HBI), as a fluorophore covalently bound to an oligonucleotide probe and potentially capable of intercalating within a probe-mRNA duplex. We first synthesized the two-ring scaffold of the HBI chromophore 5 and coupled it to 2'-deoxyuridine at C5-position via a 7-atom-spacer, to give 4. Indeed, in the highly viscous glycerol used to mimic the reduced conformational flexibility of the intercalated HBI, chromophore 4 displayed a quantum yield of 0.29 and brightness of 20600 M^-1cm^-1, while no fluorescent signal was observed in methanol. Next, we synthesized a 20-mer oligonucleotide probe incorporating 4 at position 6 (5'-CCCGTUTCAACAGGAGTTTC-3'), ON^HBI, targeting nucleotides 1233-1253 of HER-2 mRNA. A 16-fold enhancement of ON^HBI emission intensity upon hybridization with the complementary RNA vs that of the oligonucleotide probe alone indicated the presence of target oligonucleotide and proved the intercalation of the chromophore (quantum yield 0.52; brightness 23500 M^-1cm^-1). Even more, an 11-fold enhancement of ON^HBI emission (quantum yield 0.50; brightness 23200 M^-1cm^-1) was observed when the probe was mixed with total RNA extract from a human cell line that has high levels of HER2 mRNA expression. Thus, we propose ON^HBI as a promising probe potentially useful for the sensitive and specific detection of HER2 mRNA breast cancer marker.