title: Synthesis and evaluation of a photoresponsive quencher for fluorescent hybridization probes.
abstract: Nowadays, most nucleic acid detections using fluorescent probes rely on quenching of fluorescence by energy transfer from one fluorophore to another or to a non-fluorescent molecule (quencher). The most widely used quencher in fluorescent probes is 4-((4-(dimethylamino)phenyl)azo)benzoic acid (DABCYL). We targeted a nucleoside-DABCYL analogue which could be incorporated anywhere in an oligonucleotide sequence and in any number, and used as a quencher in different hybridization sensitive probes. Specifically, we introduced a 5-(4-((dimethylamino)phenyl)azo)benzene)-2'-deoxy-uridine (dU(DAB)) quencher. The photoisomerization and dU(DAB)'s ability to quench fluorescein emission have been investigated. We incorporated dU(DAB) into a series of oligonucleotide (ON) probes including strand displacement probes, labeled with both fluorescein (FAM) and dU(DAB), and TaqMan probes bearing one or two dU(DAB) and a FAM fluorophore. We used these probes for the detection of a DNA target in real-time PCR (RT-PCR). All probes showed amplification of targeted DNA. A dU(DAB) modified TaqMan RT-PCR probe was more efficient as compared to a DABCYL bearing probe (93% vs. 87%, respectively). Furthermore, dU(DAB) had a stabilizing effect on the duplex, causing an increase in Tm up to 11 °C. In addition we showed the photoisomerisation of the azobenzene moiety of dU(DAB) and the dU(DAB) triply-labeled oligonucleotide upon irradiation. These findings suggest that dU(DAB) modified probes are promising probes for gene quantification in real-time PCR detection and as photoswitchable devices.
authors.affiliation: Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel. bilha.fischer@biu.ac.il.
authors.forename: Marina
authors.initials: M
authors.lastname: Kovaliov
authors.forename: Chaim
authors.initials: C
authors.lastname: Wachtel
authors.forename: Eylon
authors.initials: E
authors.lastname: Yavin
authors.forename: Bilha
authors.initials: B
authors.lastname: Fischer
mesh_terms: D060326:Chemistry Techniques, Synthetic; D003857:Deoxyuridine / Q000737:chemistry; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry*; D007536:Isomerism; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000737:chemistry; D009943:Organophosphorus Compounds / Q000737:chemistry; D055668:Photochemical Processes; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D009841:Oligonucleotides; D009943:Organophosphorus Compounds; C434331:phosphoramidite; C063127:4-(4-dimethylaminophenylazo)benzoic acid; D004124:p-Dimethylaminoazobenzene; D003857:Deoxyuridine