title: IMAGEtags: Quantifying mRNA Transcription in Real Time with Multiaptamer Reporters.
abstract: Cell communications are essential to the organization, development, and maintenance of multicellular organisms. Much of this communication involves changes in RNA transcription and is dynamic. Most methods for studying transcription require interrupting the continuity of cellular function by sacrificing the communicating cells and capturing gene expression information by periodic sampling of individual cells or the population. The IMAGEtag technology to quantify RNA levels in living cells, demonstrated here in yeast, allows individual cells to be tracked over time as they respond to different environmental cues. IMAGEtags are short RNAs consisting of strings of a variable number of tandem aptamers that bind small-molecule ligands. The aptamer strings can vary in length and in configuration of aptamer constituents, such as to contain multiples of the same aptamer or two or more different aptamers that alternate in their occurrence. A minimum effective length is about five aptamers. The maximum length is undefined. The small-molecule ligands are enabled for imaging as fluorophore conjugates. For each IMAGEtag, two fluorophore conjugates are provided, which are FRET pairs. When a cell expresses an RNA containing an IMAGEtag sequence, the aptamers bind their ligands and bring the fluorophores into sufficiently close proximity to allow FRET. The background fluorescence of both fluorophores is minimal in the FRET channel. These features endow IMAGEtags with the sensitivity to report on mRNA expression levels in living cells.
authors.affiliation: Cornell University, Ithaca, NY, United States. Electronic address: jr839@cornell.edu.
authors.forename: J
authors.initials: J
authors.lastname: Ray
authors.affiliation: National Forensic Service, Seoul, South Korea.
authors.forename: I
authors.initials: I
authors.lastname: Shin
authors.affiliation: Aptalogic Inc., Ames, IA, United States.
authors.forename: M
authors.initials: M
authors.lastname: Ilgu
authors.affiliation: Ames Laboratory, US DOE, Ames, IA, United States; Iowa State University, Ames, IA, United States.
authors.forename: L
authors.initials: L
authors.lastname: Bendickson
authors.affiliation: The Scripps Research Institute, Jupiter, FL, United States.
authors.forename: V
authors.initials: V
authors.lastname: Gupta
authors.affiliation: Ames Laboratory, US DOE, Ames, IA, United States; Iowa State University, Ames, IA, United States.
authors.forename: G A
authors.initials: GA
authors.lastname: Kraus
authors.affiliation: Ames Laboratory, US DOE, Ames, IA, United States; Iowa State University, Ames, IA, United States; Aptalogic Inc., Ames, IA, United States. Electronic address: marit@iastate.edu.
authors.forename: M
authors.initials: M
authors.lastname: Nilsen-Hamilton
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis*; D008024:Ligands; D061848:Optical Imaging / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*; D014158:Transcription, Genetic; D015003:Yeasts / Q000235:genetics*
chemical_list: D052157:Aptamers, Nucleotide; D005456:Fluorescent Dyes; D008024:Ligands; D012333:RNA, Messenger
keywords: FRET; Fluorescence; IMAGEtags; RNA imaging; Yeast