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RNA visualization in live bacterial cells using fluorescent protein complementation

Abstract

We describe a technique for the detection and localization of RNA transcripts in living cells. The method is based on fluorescent-protein complementation regulated by the interaction of a split RNA-binding protein with its corresponding RNA aptamer. In our design, the RNA-binding protein is the eukaryotic initiation factor 4A (eIF4A). eIF4A is dissected into two fragments, and each fragment is fused to split fragments of the enhanced green fluorescent protein (EGFP). Coexpression of the two protein fusions in the presence of a transcript containing eIF4A-interacting RNA aptamer resulted in the restoration of EGFP fluorescence in Escherichia coli cells. We also applied this technique to the visualization of an aptamer-tagged mRNA and 5S ribosomal RNA (rRNA). We observed distinct spatial and temporal changes in fluorescence within single cells, reflecting the nature of the transcript.

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Figure 1: Design of RNA aptamer-based fluorescent protein complementation.
Figure 2: Protein complementation of split EGFP detects RNA in live bacterial cells.
Figure 3: Fluorescence changes within a single bacterium.
Figure 4: Changes in RNA concentration as determined by MALDI TOF MS.
Figure 5: Localization of LacZ mRNA and 5S rRNA in live bacterial cell.

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Acknowledgements

We thank A. Randhawa for technical assistance, C. Witte-Hoffmann for suggesting the use of eIF4A, C. Proud (University of Dundee, UK) for the eIF4A clone, P. Moore (Yale University) for pKK5-1 clone, S. Mazzei (Cleveland Clinic Foundation) for BD EGFP beads, V. Demidov for measuring cell fluorescence spectra and constant support, A. Gershteyn for help with preparation of figures, I. Keren and G. Balaszi for discussions and support. We thank J.J. Collins for reading the manuscript and for valuable suggestions, and all members of the Center for Advanced Biotechnology for help, insightful discussions and suggestions. This work was sponsored by Hamilton Thorne Biosciences and supported in part by a Charles E. Culpeper Biomedical Pilot Grant from Goldman Philanthropic Partnership to N.E.B.

Author information

Authors and Affiliations

Authors

Contributions

M.V.-B. performed cloning, flow cytometry and microscopy analysis, R.M. performed rcPCR and MALDI TOF analysis, C.R.C. and N.E.B. were responsible for project planning. N.E.B. drafted the paper. M.V.-B., R. M., C.R.C. and N.E.B. discussed the results and extensively revised the manuscript.

Corresponding author

Correspondence to Natalia E Broude.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Cells expressing RNA without aptamer sequence display low fluorescence. (PDF 13 kb)

Supplementary Fig. 2

Cells expressing aptamer-containing target RNA from a higher copy vector (100 copies) display higher fluorescence. (PDF 18 kb)

Supplementary Fig. 3

Comparison between fluorescence spectra of full-length EGFP and the reconstructed EGFP shows clear differences. (PDF 19 kb)

Supplementary Fig. 4

Single-cell fluorescence profiles in two independent experiments show fluctuations and synchronization of the signal over time. (PDF 21 kb)

Supplementary Methods (PDF 74 kb)

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Valencia-Burton, M., McCullough, R., Cantor, C. et al. RNA visualization in live bacterial cells using fluorescent protein complementation. Nat Methods 4, 421–427 (2007). https://doi.org/10.1038/nmeth1023

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