Expanding the Functions of KHSRP Protein: Insights into DNA G‐Quadruplex Binding

Abstract KHSRP (KH‐type splicing regulatory protein) is a multifunctional nucleic acid‐binding protein that regulates various cellular processes, with critical roles in controlling gene expression. G‐quadruplexes (G4s) are noncanonical nucleic acid structures involved in essential cellular activities, including gene expression, and are recognized as potential therapeutic targets in cancer. The biological functions of G4s are mediated by proteins making their formation highly dynamic within cells. Therefore, the recognition of G4s by specific proteins is crucial for modulating physiological and pathological pathways. Given the growing interest in DNA G4s, a deeper understanding of the proteins that interact with them and their molecular recognition is imperative. This study demonstrates that KHSRP binds to these DNA structures. Biophysical analyses provide insights into the thermodynamics, kinetics, and structural aspects of these interactions, showing that G4 structural variability significantly influences KHSRP binding, in which the KH3 protein domain plays a key role. Validation of these interactions in cancer cells further highlights their biological relevance. Notably, the G4 ligand pyridostatin affects KHSRP/G4 interactions both in vitro and in cells, suggesting that small molecules can modulate this molecular recognition. These findings underscore KHSRP's potential role in regulating cellular mechanisms through binding to G4‐forming DNA, positioning it as a possible therapeutic target in cancer.