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1 DOI: /NCHEM.1805 Visualization and Selective Chemical Targeting of RNA G-quadruplex Structures in the Cytoplasm of Human Cells Giulia Biffi 1, Marco Di Antonio 2, David Tannahill 1 and Shankar Balasubramanian 1,2 * 1 Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, 2 Cambridge, CB2 0RE, UK; 2 Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK. * sb10031@cam.ac.uk NATURE CHEMISTRY 1

2 Supplementary Figure 1: Circular dichroism (CD) spectroscopy of G-quadruplex and control oligonucleotides. Top: CD spectra signatures of control oligonucleotides that do not form a G-quadruplex structure (RNA hairpin, TERRA-lin and TERRA-mut). Middle: CD spectra signatures of TERRA, NRAS and BCL2 RNA G-quadruplex oligonucleotides. The positive peak at ~260 nm and the negative peak at ~240 nm are characteristic of a parallel G-quadruplex structure adopted by these oligonucleotides under the experimental conditions. Bottom: CD melting for TERRA, NRAS and BCL2 G-quadruplexes obtained by plotting normalized molar ellipticity recorded at 265 nm. Melting temperatures (Tm) are indicated. These results indicate the high thermal stability of the G-quadruplexes utilized in ELISA experiments. NATURE CHEMISTRY 2

3 Supplementary Figure 2: Visualization of RNA G-quadruplexes in the cytoplasm of a range of human cell types. Indirect immunofluorescence microscopy showing detection of RNA G- quadruplexes by the G-quadruplex structure-specific antibody, BG4, in the cytoplasm of a, U2OS (osteosarcoma), b, HeLa (cervical carcinoma), c, HUVEC (human umbilical vein endothelial cells) and d, GM847 (SV40 immortalized human skin fibroblasts) cells. BG4 foci are colored red by staining with Alexafluor 594 fluorescent dye. Nuclei are colored blue by counterstaining with the general DNA dye DAPI. The borders of the cytoplasm are indicated with dotted lines. Note that in a and d it was not possible to discern all cytoplasmic boundaries due to cell overlap. Scale bar in a corresponds to 20 µm. These results show the generality of RNA G-quadruplex existence in the cytoplasm of a range of human cancer and normal cell lines. NATURE CHEMISTRY 3

4 Supplementary Figure 3: Confirmation that RNA G-quadruplex structures localize to the cytoplasm of human cells. BG4 antibody staining for G-quadruplex structures is confined within the cell boundaries of MRC5 SV40 transformed fibroblasts, as assessed by colocalization with CellMask TM green stain (Life Technologies), a general plasma membrane stain. a, untreated cells. b, Cells incubated with the G-quadruplex-stabilizing ligand pyridostatin (PDS) (see Fig. 3 for the experiment rationale). PDS treatment increases the number of BG4 foci relative to the untreated control (a). c, Cells treated with RNase A before NATURE CHEMISTRY 4

5 staining with BG4. Cytoplasmic staining is lost after RNase A treatment. The left hand images show three layers superimposed: nuclei (blue), cytoplasm (green) and BG4 (red). In these images, the blue color of the nuclei is masked by the intense CellMask TM green stain. Also, BG4 foci appear as punctate yellow spots due to the overlap of red and green channels. For clarity, the right hand images show the same cells but only with the red and blue channels superimposed, thus BG4 staining is red, nuclei are blue and cytoplasm is dark. The dotted lines indicate the cytoplasmic boundaries. Nuclei are colored blue due to counterstaining with the general DNA dye DAPI. Scale bar in a corresponds to 20 µm. Supplementary Figure 4: Visualization of RNA G-quadruplex structures in the cytoplasm of human cells following fixation with ethanol. Indirect immunofluorescence microscopy showing BG4 nuclear and cytoplasmic staining (red) in MRC5 SV40 transformed fibroblasts after fixation with ethanol, a precipitating fixative. BG4 staining is seen as punctate red foci due to staining with Alexafluor 594 red fluorescent dye. Nuclei are colored blue due to counterstaining with the general DNA dye DAPI. The dotted lines indicate the cytoplasmic boundaries. This result shows that the presence of both nuclear (DNA) and cytoplasmic (RNA) G-quadruplexes following ethanol fixation is comparable to that seen with formaldehyde fixation (e.g. see Fig. 2). Scale bar corresponds to 10 µm. NATURE CHEMISTRY 5

6 Supplementary Figure 5: BG4 antibody binding for RNA G-quadruplexes is unaffected by the presence of small molecule ligands. Binding curves as determined by ELISA show that BG4 affinity for the RNA G-quadruplex BCL2 is not affected by either prior incubation with (a) PDS or (c) carboxypyridostatin (carboxypds) before BG4 addition, or by simultaneous addition of BG4 with (b) PDS or (d) carboxypds. Dissociation constants (K d ) are indicated. The error bars represent the standard error of the mean calculated from three replicates. These results are comparable with that of BG4 binding to BCL2 in the absence of small molecule ligands (Fig. 1c, K d = 6.5±0.9 nm). NATURE CHEMISTRY 6

7 Supplementary Figure 6: G-quadruplex nuclear foci are sensitive to DNase but not RNase treatment. DNA G-quadruplexes in the nuclei of MRC5 SV40 transformed fibroblasts are revealed by staining with the BG4 antibody. Nuclei are colored blue due to counterstaining with the general DNA dye DAPI. The cytoplasm surrounding the nuclei is dark. BG4 foci are red due to staining with Alexafluor 594 red fluorescent dye. BG4 nuclear NATURE CHEMISTRY 7

8 foci disappear after DNase treatment in both (a) PDS or (c) carboxypds treated cells compared to a no nuclease-treated control (Fig. 4d, e). On the contrary, BG4 nuclear foci are not affected by RNase A treatment in (b) PDS or (d) carboxypds treated cells. For clarity in (a) and (c), dotted lines indicate nuclei boundaries. Scale bar in a corresponds to 10 µm. e, Quantification of BG4 foci number per nucleus for a-d and Fig. 4d,e cells were counted per condition and the standard error of the mean calculated from three replicates. The p values for the DNase and RNase treatments relative to the no nuclease control, were calculated using the Student s t-test. The DNase-treated samples showed a highly significant reduction in BG4 foci (** p< 0.001), whereas the RNase-treated samples were not significantly different from the no nuclease control. These results reaffirm that the BG4 antibody detects DNA G-quadruplexes in cell nuclei (see also Biffi et al, 2013). NATURE CHEMISTRY 8