Fig. 3: Biophysical assays demonstrate temperature and potassium dependent G-quadruplex formation of RBM3 R1 in vitro.

a Sequences of synthesized WT and mutant rG4-R1 RNAs. R1 corresponds to the sequence near the 3′ splice site of RBM3 eoxn 3a (Fig. 2j). b A schematic of biophysical CD spectroscopy assays. The RNA samples were heated to 95 °C for 5 min and then cooled to room temperature overnight for renaturation. Subsequently, the samples were scanned from 220 to 310 nm at different temperatures. After setting the CD spectrometer to the desired measurement temperature, RNA samples were incubated at that temperature for 30 min to ensure thermal equilibrium and structural stabilization. CD spectra were then recorded using a response time of 0.5 s per nm. Each spectrum represents the average of three consecutive scans collected under these conditions. It was generated with BioRender. c Circular dichroism (CD) spectrum signal of rG4-R1 and its mutant in different potassium concentrations at 37 °C. X-axis: the wavelength of light; y-axis: the magnitude of the CD signal. Signal peak at 265 nm and 240 nm: parallel rG4 structure. d, e Normalized CD signal of rG4-R1 at different temperatures in low KCl concentration (d) and high KCl condition (e). [θ] = θ_obs × 100/(c × l), where [θ] = molar ellipticity in deg·cm²·dmol⁻¹; θ_obs = observed ellipticity in millidegrees (mdeg); c = molar concentration of the sample in mol/L; l = path length in cm; Multiply by 100 to convert mdeg to deg and account for units. f Summary of CD signal peaks at 265 nm (top) and 240 nm (bottom) of rG4-R1 at various temperatures under both high and low concentrations of KCl. g, h 1D ¹H NMR spectrum showing the imino region (g, peaks indicating structured rG4s) and aromatic protons (h, as a measure for unstructured RNA, see text for details), confirming that the rG4-R1 sequence forms a temperature-sensitive rG4 in vitro. Source data are provided as a Source Data file.