Single molecule–sensitive probes for imaging RNA in live cells

Abstract

To visualize native or non-engineered RNA in live cells with single-molecule sensitivity, we developed multiply labeled tetravalent RNA imaging probes (MTRIPs). When delivered with streptolysin O into living human epithelial cancer cells and primary chicken fibroblasts, MTRIPs allowed the accurate imaging of native mRNAs and a non-engineered viral RNA, of RNA co-localization with known RNA-binding proteins, and of RNA dynamics and interactions with stress granules.

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Figure 1: Production and imaging of MTRIPs.
Figure 2: Imaging of single molecules of β-actin mRNA in an A549 cell.
Figure 3: Transient interactions between viral genomic RNA of hRSV and GFP–TIA-1.

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Acknowledgements

This study was supported by the Georgia Institute of Technology administration (P.J.S.).

Author information

P.J.S. designed and performed experiments, analyzed data and wrote manuscript, A.W.L. performed experiments and analyzed data, P.C. performed experiments, Y.S. and G.J.B. provided novel antibody, M.E.L. and J.E.C. provided virus and antibody and edited manuscript.

Correspondence to Philip J Santangelo.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1-10, Supplementary Table 1 and Supplementary Results (PDF 5705 kb)

Supplementary Video 1

Time-lapse images of β-actin mRNA granules in live cell in one optical plane taken at 1 Hz for 3.0 minutes; first 1 minute is played back at 3 frames a second for 20 s. (MOV 2584 kb)

Supplementary Video 2

Time-lapse images of the trajectory of a single β-actin mRNA granule in a live cell in one optical plane taken at 1 Hz but played back at 3 frames a second. This movie represents only 70 s of the 3 minutes of total data taken in this cell. (MOV 3319 kb)

Supplementary Video 3

Time-lapse images of the trajectory of a single β-actin mRNA granule in a live cell in one optical plane taken at 5 Hz but played back at 3 frames a second for 30 s. (MOV 2375 kb)

Supplementary Video 4

Time-lapse images of stress granule (green) collision and penetration of a viral RNA granule (red). Images were taken every 5 seconds, and playback is at 3 frames per second. (MOV 529 kb)

Supplementary Video 5

Time-lapse images of the separation of the stress granule (green) with the viral RNA granule (red). Images were taken every 5 seconds, and playback is at 3 frames per second. (MOV 429 kb)

Supplementary Video 6

Images of the docking of a stress granule (green) and viral RNA granule (red). Images were taken every 5 seconds, and playback is at 3 frames per second. (MOV 610 kb)

Supplementary Video 7

Movie showing an example of granule fusion, where both viral RNA (red) and TIA-1 (green) are imaged. Images were taken every 5 seconds, and playback is at 3 frames per second. (MOV 1174 kb)

Supplementary Video 8

Movie showing an example of granule splitting, where both viral RNA (red) and TIA-1 (green) are imaged. Images were taken every 5 seconds, and playback is at 3 frames per second. (MOV 928 kb)

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