Supplementary Figure 9: Performance comparison of Peppers and Riboglow platform.
From: Visualizing RNA dynamics in live cells with bright and stable fluorescent RNAs
(a) Structures of Cbl-5xPEG-ATTO 590 and HBC620. Cbl-5xPEG-ATTO 590 is a macromolecule (MW>2000) that is hard to traverse plasma membrane. (b) Excitation and emission spectra of Pepper620 and Riboglow (the complex of RNA tag A and Cbl-5xPEG-ATTO 590). (c) Fluorescence activation of Cbl-5xPEG-ATTO 590 and HBC620 upon binding with their specific aptamers measured at 590 nm excitation 640 nm emission. Data represent the mean ± s.d. from three technical replicates. (d) and (e) Live cell imaging of G3BP1-BFP and ACTB-(A)4x (d) or ACTB-4Pepper (e) during stress granules (SGs) formation in cells treated with NaAsO2. (A)4x is the tandem tetramer of RNA tag A. G3BP1 protein is a granule-associated RNA-binding protein and a marker to localize SGs. Scale bar, 10 µm. (f) and (g) Line profiles of the fluorescence from G3BP1-BFP and ACTB-(A)4x (f) or ACTB-4Pepper (g) of the dashed lines in (d) and (e). (h) Schematic representation of constructs expressing 4Pepper or A(4x) alone from Pol II (U6 promoter), or mRNAs fused with 4Pepper or A(4x) tag from Pol III (CMV promoter). (i) Live cell imaging of Riboglow or Pepper620, and their fusions transcribed by Pol II or Pol III. Scale bar, 10 µm. (j) Quantitative analysis of the fluorescence of Riboglow or Pepper620, and their fusions in cells. Statistical comparison was performed by two-tailed t test; N.S., no significant difference. Data represent the mean ± s.d. (N = 50 cells). (k) Schematic representation of constructs for the targeting of 4Pepper or A(4x) fused ACTB mRNA to outer membrane of mitochondria through TOMM20-tdMCP-BFP tethering. (l) Imaging of mitochondrial localized mRNA labeled with Riboglow and Pepper620. The COS-7 cells co-transfected with TOMM20-tdMCP-BFP and ACTB-4Pepper or ACTB-(A)4x expression plasmids were incubated with HBC620 or loaded with Cbl-5xPEG-ATTO 590, respectively. Scale bar, 10 µm. (m) Quantification of the fluorescence of Pepper620, Riboglow and BFP in the outer membrane of mitochondrial and cytosol. Statistical comparison was performed by two-tailed t test. Data represent the mean ± s.d. (N = 50 cells). For b, d-g, i and l, at least two independent experiments were carried out with similar results.
