摘要：
AbstractFluorescent 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 (λem 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.Article Outline1. Introduction2. Results2.1. Design of NIF-labeled-oligonucleotide probes2.2. Incorporation of 8-(p-CF3-cinnamyl)-2′-deoxy-adenosine (6) into oligonucleotides2.3. Photophysical characterization of cyclin D1-targeting ss–NIF–probes2.4. Photophysical characterization of cyclin D1-targeting ds-NIF-probes2.5. Evaluating the ability of monomer 6 to retain the hybridization preference of adenosine2.6. Reaction kinetics with double-stranded probes and a ssDNA target2.7. Reaction kinetics with double-stranded probes and a ssRNA target2.8. Using ON7 for the application of ds-NIF-probe methodology2.9. Application of ds-NIF-probe for detection of cyclin D1 mRNA in total RNA extracts of cancerous cells2.10. Thermostability of duplexes3. Discussion4. Experimental4.1. General4.2. Extinction coefficients calculations4.3. Oligonucleotide synthesis and purification4.4. UV measurements4.5. Fluorescence measurements4.6. Quantum yield determination4.7. Hybridization of oligonucleotides4.8. Thermal denaturation measurements (Tm)4.9. Reaction kinetics of displacement hybridization4.10. Cell culture and RNA cell extract4.11. Fluorescence measurements of double-stranded probe in total cell RNA extracts4.12. Synthesis4.12.1. 5-(6-Amino-8-bromo-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl) methoxy)methyl)tetrahydrofuran-3-ol (7) [34]AbstractFluorescent nucleoside analo
