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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.

Author information

1
Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL 61801 USA.
2
Department of Materials Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801 USA.
3
Howard Hughes Medical Institute, Baltimore, MD 21205 USA.
4
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205 USA.
5
Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218 USA.
6
Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205 USA.

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.

PMID:
26612428
PMCID:
PMC4661537
DOI:
10.1038/srep17295
[Indexed for MEDLINE]
Free PMC Article

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