Single-cell imaging of individual mRNAs has revealed core mechanisms of the central dogma. However, most approaches require cell fixation or have limited sensitivity for live-cell applications. Here, we describe SunRISER (SunTag-based reporter for imaging signal-enriched mRNA), a computationally and experimentally optimized approach for unambiguous detection of single mRNA molecules in living cells. When viewed by epifluorescence microscopy, SunRISER-labeled mRNAs show strong signal to background and resistance to photobleaching, which together enable long-term mRNA imaging studies. SunRISER variants, using 8× and 10× stem-loop arrays, demonstrate effective mRNA detection while significantly reducing alterations to target mRNA sequences. We characterize SunRISER to observe mRNA inheritance during mitosis and find that stressors enhance diversity among post-mitotic sister cells. Taken together, SunRISER enables a glimpse into living cells to observe aspects of the central dogma and the role of mRNAs in rare and dynamical trafficking events.
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| http://dx.doi.org/10.1016/j.crmeth.2022.100226 | DOI Listing |
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