Development of fluorescent double-strand probes labeled with 8-(p-CF ₃-cinnamyl)-adenosine for the detection of cyclin D1 breast cancer marker

Fluorescent nucleoside analogs replacing natural DNA bases in an oligonucleotide have been widely used for the detection of genetic material. Previously, we have described 2-((4-(trifluoromethyl) phenyl)-trans-vinyl)- 2′-deoxy-adenosine, 6, a nucleoside analog with intrinsic fluorescence (NIF). Analog 6 exhibits a quantum yield 3115-fold higher than that of adenosine (φ 0.81) and maximum emission which is 120 nm red shifted (λ_em 439 nm). Here, we incorporated this analog in one or several positions of cyclin D1-targeting 15-mer oligonucleotides (ONs). The fluorescence of 6 was quenched upon incorporation into an oligonucleotide (ca. 1.5-22 fold), and was further reduced upon duplex formation. Specifically, ON7 exhibited a fluorescence decrease of ca. 2- or 3-fold upon duplex formation with complementary DNA or RNA strand, respectively. We determined the kinetics of dehybridization/rehybridization process in the presence of ssDNA or ssRNA targets to optimize our probes length and established the probes' selectivity towards a specific target. Furthermore, we proved specificity of our probe to the target vs. singly mismatched targets. Our most promising ds-NIF-probe, ON7:RNA, was used for the detection of cyclin D1 mRNA marker in cancerous cells total RNA extracts. The ds-probe specifically recognized the target as observed by a 2-fold fluorescence increase within 30 s at RT. These findings illustrate the potential of ds-NIF-probes for the diagnosis of breast cancer.