title: Real time monitoring of endogenous cytoplasmic mRNA using linear antisense 2'-O-methyl RNA probes in living cells.
abstract: Visualization and monitoring of endogenous mRNA in the cytoplasm of living cells promises a significant comprehension of refined post-transcriptional regulation. Fluorescently labeled linear antisense oligonucleotides can bind to natural mRNA in a sequence-specific way and, therefore, provide a powerful tool in probing endogenous mRNA. Here, we investigated the feasibility of using linear antisense probes to monitor the variable and dynamic expression of endogenous cytoplasmic mRNAs. Two linear antisense 2'-O-methyl RNA probes, which have different interactive fluorophores at the 5'-end of one probe and at the 3'-end of the other, were used to allow fluorescence resonance energy transfer (FRET) upon hybridization to the target mRNA. By characterizing the formation of the probe-mRNA hybrids in living cells, we found that the probe composition and concentration are crucial parameters in the visualization of endogenous mRNA with high specificity. Furthermore, rapid hybridization (within 1 min) of the linear antisense probe enabled us to visualize dynamic processes of endogenous c-fos mRNA, such as fast elevation of levels after gene induction and the localization of c-fos mRNA in stress granules in response to cellular stress. Thus, our approach provides a basis for real time monitoring of endogenous cytoplasmic mRNA in living cells.
authors.affiliation: Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Kohki
authors.initials: K
authors.lastname: Okabe
authors.forename: Yoshie
authors.initials: Y
authors.lastname: Harada
authors.forename: Junwei
authors.initials: J
authors.lastname: Zhang
authors.forename: Hisashi
authors.initials: H
authors.lastname: Tadakuma
authors.forename: Tokio
authors.initials: T
authors.lastname: Tani
authors.forename: Takashi
authors.initials: T
authors.lastname: Funatsu
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000737:chemistry*; D003594:Cytoplasmic Granules / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D007700:Kinetics; D008856:Microscopy, Fluorescence; D016760:Proto-Oncogene Proteins c-fos / Q000032:analysis / Q000235:genetics; D015347:RNA Probes / Q000737:chemistry*; D020871:RNA Stability; D016372:RNA, Antisense / Q000737:chemistry*; D012333:RNA, Messenger / Q000032:analysis* / Q000096:biosynthesis
chemical_list: D005456:Fluorescent Dyes; D016760:Proto-Oncogene Proteins c-fos; D015347:RNA Probes; D016372:RNA, Antisense; D012333:RNA, Messenger