Fig. 5: Application of coPepper for imaging and biochemical enrichment of RNA.
From: Engineering covalent small molecule–RNA complexes in living cells
a, MsOc3HBC elicits washout-resistant Pepper fluorescence in live-cell imaging. Top panels show cells transfected or not (Mock) with pTornado-Pepper and incubated with either HBC5 (left) or MsOc3HBC (right). Bottom panels, cells were washed before imaging. Green, FLAP fluorescence; blue, Hoechst staining of DNA. Arrow designates nuclear speckles. Scale bars, 10 µm. Representative images of five independent experiments. b, MsOc3HBC is suitable for super-resolution microscopy by STED (top) and Airyscan (ASC; bottom). Circular Pepper appears at the surface of nuclear spherical structures. Panels show projections of one (top left), three (bottom left) or ten (bottom right) stacks or a maximum intensity projection rendering of a complete set of stacks (top right) generated using Huygens Professional Software. Scale bars, 2 µm. Representative images for MsOc3HBC with (W) or without washing (NW) from two (STED) or four (Airyscan) independent experiments. c, coPepper is uniquely suited for FRAP analysis. FRAP was performed on cells incubated with HBC or MsOc3HBC. Images of nuclear speckles were acquired at 0.2-s intervals before (prebleach), during (bleach) and after photobleaching. The bleached area is designated by a square. Selected frames are shown. Three independent experiments were performed. d, Quantification of FRAP measurements. Mean ± s.d. of 20 different speckles is shown. Scale bar, 5 µm. e, The bifunctional MsOc3HBC-vinyl ligand enables affinity pull-down of Pepper-containing RNA. Total RNA from HEK293T cells transfected with pTornado-Pepper was incubated with MsOc3HBC-vinyl followed by tetrazine-mediated attachment of desthiobiotin and incubation with streptavidin beads. f, Aliquots (2%) of input and flowthrough and the elution fraction (50%) were separated on a 10% TBE-urea-PA gel. Pepper RNA was visualized by direct fluorescence detection (filter for λ 450 to 495 nm) (right panel). Total RNA was stained with EtBr (left panel). Representative images of three independent experiments are shown.
