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. 2021 Mar 19;4:378. doi: 10.1038/s42003-021-01896-7

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a Schematic comparation of three single-molecule imaging methods of mRNAs in living cells. ① mRNA labeling using 24×MS2 hairpin repeats and full-length fluorescent protein fusion with tdMCP (or MCP). ② mRNA labeling using 12×MS2_PP7 hybrid hairpin repeats and split fluorescent protein fusion with MCP and PCP. ③ mRNA labeling using 1×MS2_PP7 hybrid hairpin repeat and BiFC-Tag fusion with MCP and PCP. The estimation of the labeling tag length and binding protein mass are shown. b Representative fluorescence images of single CFP mRNA labeling using 1×MS2-PP7/TagBiFC (left) or 24×PP7 (right). The 1×MS2-PP7 or 24×PP7 sequence was inserted in the 3’ UTR of coding sequence of CFP. tdPCP-HaloTag C58+HaloTag N58-tdMCP or tdPCP-EGFP was transfected to label the mRNA. Inset shows single-molecule signal from a high magnification of the box region. Scale bar, 5 μm. Inset scale bar, 1 μm. c Representative fluorescence images of single CFP mRNA 3D projection labeled using TagBiFC and single-molecule FISH. The mRNAs were counted using the Spots function of Imaris. Scale bar, 5 μm. d Quantification of the colocalization of TagBiFC and FISH signal in multiple cells. Inset shows the false-positive and false-negative ratio of TagBiFC for mRNA labeling. e Single-molecule tracking trajectory of CFP mRNA labeled by 1×MS2-PP7 or 24×PP7. Insets show single-molecule trajectories for a high magnification. f Mean square displacement of CFP mRNA labeled by 1×MS2-PP7 or 24×PP7. Data are displayed as mean ± standard deviation. g Histogram of diffusion coefficient of CFP mRNA labeled by 1×MS2-PP7 or 24×PP7. h Schematic of plasmid used for inducible reporter mRNA expression. Two expression cascades were cloned into one plasmid to ensure the co-appearance in the same cells. The rtTA was driven by EF1a promoter while CFP-MS2-PP7 was driven by tetracycline responsible element (TRE) promoter. i Representative fluorescence images of single CFP mRNA 3D projection labeled using TagBiFC in the absence or presence of 10 μg/mL tetracycline. The mRNAs were counted using the Spots function of Imaris. Scale bar, 5 μm. j CFP mRNA copy number per cell in the absence or presence of tetracycline. ***Statistically significant difference (p value = 2.193e−06, paired t test, n = 16 for the +Dox group and n = 23 for the −Dox group).