Protein purification and preparation of RNA samples

The DNA sequences encoding UP1 (residues 1–196) and UP1+RGG (residues 1–249) of hnRNPA1 (Uniprot identifier P09651-2) were sub cloned into pET28a E. coli expression vector. Both the constructs contain a C-terminal hexa-histidine (6x His) affinity purification tag. The proteins were expressed in BL21 Rosetta (DE3) cells in LB media. The cells were induced with 0.5 mM IPTG and incubated for overnight at 30°C after growing them at 37°C till the culture reached an optical density (OD) at 600 nm of 0.6. First step of purification involved 6x His-tag–Ni-NTA affinity chromatography followed by cation exchange and size exclusion chromatography (SEC) using a Superdex 75 column. The isolated RGG-box was sub cloned into pGEX-6P-1 vector and a TEV cleavage site was introduced between the GST tag and the protein. Glutathione-sepharose column was used for the purification of RGG-box, followed by the GST tag removal using TEV protease. SEC using Superdex 75 column was performed at the final step of purification of the RGG-box. The final purity of all the proteins was ascertained using SDS-PAGE and MALDI-MS analyses and the proteins were adjudged to be >95% purified.

The RNA sequences were ordered from Eurofins (Supplementary Table S1). For ITC and CD experiments the RNA samples were prepared in 20 mM Tris, pH 8.0 and 100 mM KCl. For the NMR titration experiments 10 mM potassium phosphate (pH 6.5) and 100 mM KCl was used as the buffer composition to prepare the RNA samples. The RNA samples were heated at 95°C for 3 min and then cooled at 4°C for overnight. The concentration of the RNA samples was calculated by ultraviolet (UV) absorbance at 260 nm using Eppendorf Spectrophotometer. The formation of the G-quadruplex by 6mer TERRA-6, 12mer TERRA-12, 24mer TERRA-24 RNA and 24mer abasicloopsTERRA-24 was confirmed by recording spectral scan and thermal melting using CD spectroscopy as well as by the presence of the imino proton peaks in the 1D ¹H NMR spectra.