title: Thioflavin T as a fluorescence probe for monitoring RNA metabolism at molecular and cellular levels.
abstract: The intrinsically stochastic dynamics of mRNA metabolism have important consequences on gene regulation and non-genetic cell-to-cell variability; however, no generally applicable methods exist for studying such stochastic processes quantitatively. Here, we describe the use of the amyloid-binding probe Thioflavin T (ThT) for monitoring RNA metabolism in vitro and in vivo. ThT fluoresced strongly in complex with bacterial total RNA than with genomic DNA. ThT bound purine oligoribonucleotides preferentially over pyrimidine oligoribonucleotides and oligodeoxyribonucleotides. This property enabled quantitative real-time monitoring of poly(A) synthesis and phosphorolysis by polyribonucleotide phosphorylase in vitro. Cellular analyses, in combination with genetic approaches and the transcription-inhibitor rifampicin treatment, demonstrated that ThT mainly stained mRNA in actively dividing Escherichia coli cells. ThT also facilitated mRNA metabolism profiling at the single-cell level in diverse bacteria. Furthermore, ThT can also be used to visualise transitions between non-persister and persister cell states, a phenomenon of isogenic subpopulations of antibiotic-sensitive bacteria that acquire tolerance to multiple antibiotics due to stochastically induced dormant states. Collectively, these results suggest that probing mRNA dynamics with ThT is a broadly applicable approach ranging from the molecular level to the single-cell level.
authors.affiliation: Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Chuo-Ku, Kumamoto 860-0811, Japan Department of Bacteriology, The Jikei University School of Medicine, Minato-Ku, Tokyo 105-8461, Japan ssugimoto@jikei.ac.jp.
authors.forename: Shinya
authors.initials: S
authors.lastname: Sugimoto
authors.affiliation: Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Chuo-Ku, Kumamoto 860-0811, Japan.
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Arita-Morioka
authors.affiliation: Department of Bacteriology, The Jikei University School of Medicine, Minato-Ku, Tokyo 105-8461, Japan.
authors.forename: Yoshimitsu
authors.initials: Y
authors.lastname: Mizunoe
authors.affiliation: Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Chuo-Ku, Kumamoto 860-0811, Japan.
authors.forename: Kunitoshi
authors.initials: K
authors.lastname: Yamanaka
authors.affiliation: Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Chuo-Ku, Kumamoto 860-0811, Japan ogura@gpo.kumamoto-u.ac.jp.
authors.forename: Teru
authors.initials: T
authors.lastname: Ogura
mesh_terms: D000244:Adenosine Diphosphate / Q000032:analysis; D052160:Benzothiazoles; D005456:Fluorescent Dyes; D011117:Polyribonucleotide Nucleotidyltransferase / Q000032:analysis / Q000378:metabolism; D012313:RNA / Q000378:metabolism*; D012329:RNA, Bacterial / Q000378:metabolism; D059010:Single-Cell Analysis; D013844:Thiazoles / Q000378:metabolism
chemical_list: D052160:Benzothiazoles; D005456:Fluorescent Dyes; D012329:RNA, Bacterial; D013844:Thiazoles; C009462:thioflavin T; D000244:Adenosine Diphosphate; D012313:RNA; D011117:Polyribonucleotide Nucleotidyltransferase