Fig. 4: Intracellular tethering of Pepper and preQ1 RNA.
From: Engineering covalent small molecule–RNA complexes in living cells
a, Schematic depiction of the experimental setup. HEK293T cells were transfected with pTornado-Pepper (left) or pTornado-preQ1-Pepper (right), grown to enable expression of the circular pepper aptamer and then incubated with either HBC or MsOc3HBC (left) or Brc3preQ1 (right), followed by total RNA extraction, PAGE and fluorescence detection or primer extension. In other experiments, live-cell imaging with or without washing with PBS was performed. b, PAGE analysis shows that MsOc3HBC forms a covalent tether intracellularly and remains stably attached to the RNA during RNA extraction under denaturing conditions. Left panel, coPepper aptamers allow for direct fluorescence detection by a Typhoon imager: the fluorescent bands (first and second lane) correspond to the circular Pepper and preQ1-Pepper RNAs, respectively. Middle panel, gel was stained with HBC and imaged again: circular RNAs without covalently attached fluorophores become visible (fourth and fifth lane). Right panel, gel was stained with ethidium bromide (EtBr) to visualize total RNA. M, Low Range single-stranded RNA ladder (NEB). Representative image of three independent experiments is shown. HBC was tested once. c, Brc3preQ1 and Brc3DPQ1 cause premature abortion of primer extension adjacent to the alkylated G5 (highlighted in yellow) when crosslinked to their cognate aptamers. Transfected cells were incubated with different concentrations of Brc3preQ1 and Brc3DPQ1 as indicated, and primer extension was performed with isolated total RNA (primer sequence in gray). Sequencing ladders (C,T,A,G) were generated by reverse transcription of total RNA (not incubated with ligand) and addition of the corresponding dideoxynucleotides. Representative images of four (Brc3preQ1) or two (Brc3DPQ1) independent experiments are shown.
