Fig. 4. Targeted validation of RNA G4s found in splicing sites in presence of a G4 stabilising cation (K⁺) and a non-G4 stabilising cation (Li⁺).

A UCSC screenshot displaying the SLC6A17 locus and the distribution of G4 consensus motifs, G4-seq derived G4 peaks and protein domains. B–D Circular dichroism (CD) spectra of the three candidate targets for RNA G4 formation potential in presence of two cations. The monovalent ion-dependent nature (G4 stabilised in K⁺ but not in Li⁺) and the CD profile (positive peak at 262 nm and negative peak at 240 nm) indicate the formation of RNA G4s with parallel topology. E, F UV-melting profiles of the three G4 candidates in presence of Li⁺ and K⁺. Hyperchromic shift at 295 nm is a hallmark for G4 formation, which can be transformed to a negative peak in the derivative plot (dAbs/dT) for G4 stability analysis. The melting temperature (Tm) of a G4 can be identified at the maximum negative value. For the K⁺ treatment, the Tms of the RNA G4 are >85 °C. G–I Fluorescence emission associated with NMM ligand binding to RNA G4 candidates in the presence of Li⁺ or K⁺ ions^. In the absence of NMM ligand, no fluorescence was observed at ~610 nm. Upon NMM addition, weak fluorescence was observed under Li⁺, which was substantially enhanced when substituted with K⁺, supporting the formation of RNA G4 which allows recognition of NMM and enhances its fluorescence.