title: Detection of cyclin D1 mRNA by hybridization sensitive NIC-oligonucleotide probe.
abstract: A large group of fluorescent hybridization probes, includes intercalating dyes for example thiazole orange (TO). Usually TO is coupled to nucleic acids post-synthetically which severely limits its use. Here, we have developed a phosphoramidite monomer, 10, and prepared a 2'-OMe-RNA probe, labeled with 5-(trans-N-hexen-1-yl-)-TO-2'-deoxy-uridine nucleoside, dU(TO), (Nucleoside bearing an Inter-Calating moiety, NIC), for selective mRNA detection. We investigated a series of 15-mer 2'-OMe-RNA probes, targeting the cyclin D1 mRNA, containing one or several dU(TO) at various positions. dU(TO)-2'-OMe-RNA exhibited up to 7-fold enhancement of TO emission intensity upon hybridization with the complementary RNA versus that of the oligomer alone. This NIC-probe was applied for the specific detection of a very small amount of a breast cancer marker, cyclin D1 mRNA, in total RNA extract from cancerous cells (250 ng/μl). Furthermore, this NIC-probe was found to be superior to our related NIF (Nucleoside with Intrinsic Fluorescence)-probe which could detect cyclin D1 mRNA target only at high concentrations (1840 ng/μl). Additionally, dU(T) can be used as a monomer in solid-phase oligonucleotide synthesis, thus avoiding the need for post-synthetic modification of oligonucleotide probes. Hence, we propose dU(TO) oligonucleotides, as hybridization probes for the detection of specific RNA in homogeneous solutions and for the diagnosis of breast cancer.
authors.affiliation: Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel.
authors.forename: Marina
authors.initials: M
authors.lastname: Kovaliov
authors.affiliation: School of Pharmacy, Institute for Drug Research, The Hebrew University of Jerusalem, Ein Karem, Jerusalem 91120, Israel.
authors.forename: Meirav
authors.initials: M
authors.lastname: Segal
authors.affiliation: Faculty of Life Sciences and Institute of Nanotechnology, Bar-Ilan University, Ramat-Gan 52900, Israel.
authors.forename: Pinhas
authors.initials: P
authors.lastname: Kafri
authors.affiliation: School of Pharmacy, Institute for Drug Research, The Hebrew University of Jerusalem, Ein Karem, Jerusalem 91120, Israel.
authors.forename: Eylon
authors.initials: E
authors.lastname: Yavin
authors.affiliation: Faculty of Life Sciences and Institute of Nanotechnology, Bar-Ilan University, Ramat-Gan 52900, Israel.
authors.forename: Yaron
authors.initials: Y
authors.lastname: Shav-Tal
authors.affiliation: Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel. Electronic address: Bilha.Fischer@biu.ac.il.
authors.forename: Bilha
authors.initials: B
authors.lastname: Fischer
mesh_terms: D020137:Base Pair Mismatch; D045744:Cell Line, Tumor; D019938:Cyclin D1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D003857:Deoxyuridine / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis*; D013050:Spectrometry, Fluorescence
chemical_list: D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D019938:Cyclin D1; D003857:Deoxyuridine
keywords: Breast cancer; Cyclin D1; Fluorescent probe; RNA probe; mRNA diagnosis