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Sci Rep. 2015 Nov 27;5:17295. doi: 10.1038/srep17295.

Tandem Spinach Array for mRNA Imaging in Living Bacterial Cells.

Zhang J1,2, Fei J1, Leslie BJ1,3, Han KY1,3, Kuhlman TE1, Ha T1,4,5,6,3.

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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.
Howard Hughes Medical Institute, Baltimore, MD 21205 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.


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.

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