NMR-based conformational analysis of DNA G-quadruplex guides mapping essential structure–function relationship in protein chaperoning

G-quadruplexes (G4s) are increasingly recognized to chaperone proteins, warranting studies of structure-function relationships. In this study, we apply solution NMR methods to determine the topology and base-level resolution structure of a G-rich DNA sequence with protein chaperoning activity (referred to as Seq576) without chemical shift assignments. Seq576 samples two conformations, in the slow exchange timescale, arising from a G-register shift. Using the structural insights of Seq576, we then perform structure-function studies <i>via</i> mutation and chaperone assays to investigate the G4 properties essential for chaperoning protein aggregation and folding. These studies highlight the possibility of using a construct design to perform in-depth nucleic acid structural biology investigation using inexpensive and fast NMR experiments to obtain and analyze function, such as the residue-level investigation of chaperone activity.