Are Traditional mutant controls sufficient to identify true RNA G-quadruplex binding proteins?

G-quadruplexes (G4s) are stable non-canonical RNA structures involved in various regulatory processes, whose recognition by G4-binding proteins (G4BPs) is often studied using affinity purification and biochemical validation. A critical factor in validating true G4BPs lies in the choice of negative controls, which are frequently limited to simple G-to-A point mutations. Here, we reassess the classification of Guanine Nucleotide-Binding Protein-Like 1 (GNL1), previously identified as an RNA G4BP (rG4BP), by employing 7-deazaguanine (7dG) substitutions and salt variation strategies. Using fluorescence assays with N-methyl mesoporphyrin IX (NMM) and electrophoretic mobility shift assays (EMSAs), we show that GNL1 binds wild-type and 7dG-modified RNAs with comparable affinities. Additional experiments using potassium and lithium ions further that GNL1 binding is independent of G4 structure. Finally, truncation of PRKN RNA to its G4 core significantly reduced GNL1 binding, indicating that the protein does not interact with the folded G4 itself. These results collectively demonstrate that GNL1 binds guanine-containing sequences rather than true G4s, and that its prior classification as a G4BP was likely due to insufficient controls. Our findings highlight the importance of using robust negative controls, such as 7dG substitution and ionic condition modulation, in the accurate identification of bona fide G4BPs.