title: Tandem Spinach Array for mRNA Imaging in Living Bacterial Cells.
abstract: Live cell RNA imaging using genetically encoded fluorescent labels is an important tool for monitoring RNA activities. A recently reported RNA aptamer-fluorogen system, the Spinach, in which an RNA aptamer binds and induces the fluorescence of a GFP-like 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) ligand, can be readily tagged to the RNA of interest. Although the aptamer-fluorogen system is sufficient for imaging highly abundant non-coding RNAs (tRNAs, rRNAs, etc.), it performs poorly for mRNA imaging due to low brightness. In addition, whether the aptamer-fluorogen system may perturb the native RNA characteristics has not been systematically characterized at the levels of RNA transcription, translation and degradation. To increase the brightness of these aptamer-fluorogen systems, we constructed and tested tandem arrays containing multiple Spinach aptamers (8-64 aptamer repeats). Such arrays enhanced the brightness of the tagged mRNA molecules by up to ~17 fold in living cells. Strong laser excitation with pulsed illumination further increased the imaging sensitivity of Spinach array-tagged RNAs. Moreover, transcriptional fusion to the Spinach array did not affect mRNA transcription, translation or degradation, indicating that aptamer arrays might be a generalizable labeling method for high-performance and low-perturbation live cell RNA imaging.
authors.affiliation: Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL 61801 USA.|Department of Materials Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801 USA.
authors.forename: Jichuan
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL 61801 USA.
authors.forename: Jingyi
authors.initials: J
authors.lastname: Fei
authors.affiliation: Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL 61801 USA.|Howard Hughes Medical Institute, Baltimore, MD 21205 USA.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Leslie
authors.affiliation: Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL 61801 USA.|Howard Hughes Medical Institute, Baltimore, MD 21205 USA.
authors.forename: Kyu Young
authors.initials: KY
authors.lastname: Han
authors.affiliation: Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL 61801 USA.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Kuhlman
authors.affiliation: Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL 61801 USA.|Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205 USA.|Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218 USA.|Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205 USA.|Howard Hughes Medical Institute, Baltimore, MD 21205 USA.
authors.forename: Taekjip
authors.initials: T
authors.lastname: Ha
mesh_terms: D052157:Aptamers, Nucleotide / Q000138:chemical synthesis / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D007763:Lac Operon; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D057054:Molecular Imaging / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D018724:Spinacia oleracea / Q000737:chemistry* / Q000378:metabolism; D020080:Tandem Repeat Sequences; D014158:Transcription, Genetic; D000097573:Red Fluorescent Protein
chemical_list: D052157:Aptamers, Nucleotide; D004259:DNA-Directed DNA Polymerase; D008164:Luminescent Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D000097573:Red Fluorescent Protein; C034970:bacteriophage T7 induced DNA polymerase