Journal: STAR Protocols

Article Title: Label and quantify mRNA molecules in live cell experiments using SunRISER and dNEMO

doi: 10.1016/j.xpro.2022.101630

Figure Lengend Snippet: SunRISER plasmid variants

Article Snippet: Variation 2 – SRv.1-2P , Phage-cmv-CFP-24×pp7 (Addgene #40652) , cmv-sfgfp-gb1-scAB-ubc-nls-pcp-5×SunTag (Addgene #185800) , .

Techniques: Plasmid Preparation

Journal: Cell Reports Methods

Article Title: Long-term imaging of individual mRNA molecules in living cells

doi: 10.1016/j.crmeth.2022.100226

Figure Lengend Snippet: Design of SunRISER, a SunTag-based reporter for imaging signal-enriched mRNAs (A) Design schematic of molecular components for the SunRISER strategy to image single mRNA molecules. An mRNA transcript (black) tagged at 3′ UTR with PP7 stem loops (blue) is bound by the PCP coat protein (yellow), which is fused to a SunTag GCN4 peptide array (orange). SunTag recruits GFP (green) through antibody-peptide-specific binding between scFv (gray) and GCN4 epitopes. (B) Maximum intensity projection of z stacks for representative HeLa cells 24 h after transfection with three plasmids: phage-cmv-cfp-24×pp7, cmv-24×SunTag-PCP, and cmv-sfGFP-GB1-scAB. Scale bar: 10 μm. (C) Parameter sweeps in the space spanned by number of molecules for scFv-GFP and 24×SunTag-PCP (left) using a computational model to calculate number of GFP molecules per mRNA. Parameter combinations 1–10 were selected to represent different ratios between scFv-GFP and 24×SunTag-PCP (solid line 1:1, dotted line 5:1, and dash-dotted line 1:5), and different expression levels and frequency plots are shown (right). Different concentrations of mRNAs per cell are distinguished by different colors. See also Figures S1 and and Table S2 for binding constants.

Article Snippet: cmv-sfgfp-gb1-scAB-ubc-nls-pcp-10×SunTag (SRv.1.1-2P) , This paper , Addgene #185801.

Techniques: Imaging, Peptide Microarray, Binding Assay, Transfection, Expressing

Journal: Cell Reports Methods

Article Title: Long-term imaging of individual mRNA molecules in living cells

doi: 10.1016/j.crmeth.2022.100226

Figure Lengend Snippet: The optimal SunRISER design allows long-term imaging of single mRNA molecules throughout the cell (A) Maximum intensity projections of HeLa cells transfected with SunRISER, consisting of the detection plasmid phage-cmv-cfp-24×pp7, as well as ubc-nls-PCP-5×SunTag and cmv-sfGFP-GB1-scAB. Cells were imaged by 60× widefield fluorescence microscopy for 8 h with a 10 min framerate. Scale bar: 10 μm. See also . (B) Histograms of signal intensity and signal-to-background ratio of SunRISER-labeled single mRNAs quantified with dNEMO ( Kowalczyk et al., 2021 ). Signal intensity is defined as the average of background-corrected pixel values within the area of each detected spot. Signal-to-background ratio is calculated as the ratio of average pixel intensity within an mRNA spot divided by the average intensity of background pixels in an annular ring surrounding the spot (n = 23 for cell numbers and n = 5,611 for spots numbers). (C) Maximum intensity projection images of smFISH performed with probes against the PP7 stem loops in HeLa cells transfected with SunRISER protein components (ubc-nls-PCP-5×SunTag and cmv-sfGFP-GB1-scAB) only (top) or complete SunRISER with detection plasmid (bottom). Scale bar: 10 μm. See also Figures S2–S4 . (D) Histograms of signal intensity and signal-to-background ratio for single mRNAs labeled with smFISH Q670 probes against the mCherry coding sequence in cells expressing phage-cmv-mCherry-24×pp7 detection plasmid without (blue, n = 10 for cell numbers and n = 2,156 for spots numbers) and with (orange, n = 11 for cell numbers and n = 2,842 for spots numbers) remaining SunRISER components.

Article Snippet: cmv-sfgfp-gb1-scAB-ubc-nls-pcp-10×SunTag (SRv.1.1-2P) , This paper , Addgene #185801.

Techniques: Imaging, Transfection, Plasmid Preparation, Fluorescence, Microscopy, Labeling, Sequencing, Expressing

Journal: Cell Reports Methods

Article Title: Long-term imaging of individual mRNA molecules in living cells

doi: 10.1016/j.crmeth.2022.100226

Figure Lengend Snippet: Design principles of SunRISER are generalizable to orthogonal stem loops and protein-tagging systems (A) HeLa cells transfected with detection plasmids phage-cmv-CFP-24×MS2 (top) and phage-cmv-CFP-24×MS2V6 (bottom) stem-loop variants of SunRISER with ubc-nls-MCP-5×SunTag show similar characteristics and intensity distributions, quantified in histograms (right, top: n = 30 for cell numbers and n = 8,227 for spots numbers; bottom: n = 21 for cell numbers and n = 4,627 for spots numbers). (B) HeLa cells transfected with detection plasmid phage-cmv-cfp-24×pp7 with cmv-sfGFP-GB1-Nb-gp41 and ubc-nls-PCP-12×MoonTag, quantified in histograms (right, n = 19 for cell numbers and n = 4,534 for spots numbers). We note that the MoonRISER example can be further optimized as it uses a longer 12× MoonTag and a nanobody that has different binding properties. Cells were imaged with 60× wide-field microscope 24 h after transfection and quantified with dNEMO ( Kowalczyk et al., 2021 ) Scale bar: 10 μm. See also Figure S5 .

Article Snippet: cmv-sfgfp-gb1-scAB-ubc-nls-pcp-10×SunTag (SRv.1.1-2P) , This paper , Addgene #185801.

Techniques: Transfection, Plasmid Preparation, Binding Assay, Microscopy

Journal: Cell Reports Methods

Article Title: Long-term imaging of individual mRNA molecules in living cells

doi: 10.1016/j.crmeth.2022.100226

Figure Lengend Snippet: SunRISER is resistant to photobleaching and robustly labels mRNA molecules during prolonged imaging conditions (A) Maximum intensity projections of SunRISER SRv.1-labeled mRNAs at different time points. Twenty-four h after transfection of HeLa cells with detection plasmid phage-cmv-cfp-24×pp7 with ubc-nls-PCP-5×SunTag and cmv-sfGFP-GB1-scAB (SunRISER), cells were imaged by 60× wide-field fluorescence microscopy as 4× frame z stacks at 2 s intervals over a 1 h duration. Scale bar: 10 μm. (B) Time course of normalized signal intensity (top) and signal-to-background values (bottom) for spots labeled with SunRISER across different single cells (n = 14). Solid line marks the mean signal intensity and mean signal-to-background values averaged among single cells (signal intensity was normalized by the average spot intensity across cells at the first frame of imaging), and shaded area indicates the standard deviation between single cells. (C) Time course of mRNA counts for 1 h fast movies across different cells. The counts were smoothed by a sliding window of 5 frames. See also Figures S6 and .

Article Snippet: cmv-sfgfp-gb1-scAB-ubc-nls-pcp-10×SunTag (SRv.1.1-2P) , This paper , Addgene #185801.

Techniques: Imaging, Labeling, Transfection, Plasmid Preparation, Fluorescence, Microscopy, Standard Deviation

Journal: Cell Reports Methods

Article Title: Long-term imaging of individual mRNA molecules in living cells

doi: 10.1016/j.crmeth.2022.100226

Figure Lengend Snippet: Design of SunRISER variants (A) Scatterplot of variance and median for signal-to-background ratio of single mRNAs labeled with different combinations of PP7 stem-loop and SunTag arrays. Vertical and horizontal lines mark the preferred region in the bottom-right quadrant with small inter-spot variance and strong signal. Optimized SunRISER SRv.1 is indicated with a star. (B) Boxplots for mRNA numbers detected by different combinations of PP7 stem-loop and SunTag arrays. Gray region marks the first and third quantiles for mRNA numbers detected by optimal SunRISER SRv.1. Configuration with significantly different mRNA numbers from SRv.1 are indicated with red stars (p < 0.05, two-tailed t test). (C) Diagram of single plasmid encoding 2 protein components (C1) cmv-sfGFP-GB1-scAB and (C2) ubc-nls-PCP-5×SunTag for the SunRISER SRv.1-2P variant. (D and E) Histograms of signal-to-background ratio (D) and boxplots of mRNA numbers (E) in cells labeled with SunRISER variants SRv.1 (n = 26 for cell numbers and n = 8,713 for spots numbers) and SRv.1-2P (n = 23 for cell numbers and n = 8,875 for spots numbers). See also Figure S8 .

Article Snippet: cmv-sfgfp-gb1-scAB-ubc-nls-pcp-10×SunTag (SRv.1.1-2P) , This paper , Addgene #185801.

Techniques: Labeling, Two Tailed Test, Plasmid Preparation, Variant Assay

Journal: Cell Reports Methods

Article Title: Long-term imaging of individual mRNA molecules in living cells

doi: 10.1016/j.crmeth.2022.100226

Figure Lengend Snippet:

Article Snippet: cmv-sfgfp-gb1-scAB-ubc-nls-pcp-10×SunTag (SRv.1.1-2P) , This paper , Addgene #185801.

Techniques: Recombinant, Software