Molecular dynamics simulations elucidate the structural determinants of size-exclusion chromatography behavior in...

Research on G-quadruplexes (G4s)-RNA helicase associated with AU-rich element (RHAU) interaction has facilitated G4s-targeted therapeutic development. Despite this progress, the interaction between dimeric G-quadruplexes (d-G4s) and RHAU remain less explored compared to monomeric structures. Developing convenient and visual methods to elucidate the interaction mechanisms between different d-G4s structures and RHAU can provide new insights into binding modes, thus aiding in the design of molecular tools targeting d-G4s. In this study, we combined molecular dynamics (MD) simulations with size-exclusion chromatography (SEC) and isothermal titration calorimetry (ITC) to investigate structural dependencies in d-G4s-RHAU interactions for the first time. Diverse d-G4s included hybrid non-parallel (d-24TTG), intramolecular tandem parallel (dAGRO100, GGA8), interlocking parallel (93del), and intermolecular stacked parallel (T30695, T30177) structures were analyzed. MD simulations revealed that the distinct SEC retention behaviors in d-G4s-RHAU interactions were mainly governed by binding site accessibility, inter-site steric hindrance, and binding free energy gradients, trends supported by ITC measured affinities. Furthermore, energy decomposition analysis identified Glu26 in RHAU as a critical residue contributing to electrostatic interactions. Mutating to Arg26 substantially decreased the binding free energy (-94.05 ± 0.41 kJ/mol), emphasizing its functional importance. Thus, this work demonstrates that MD simulations are indispensable for revealing the causes of experimental phenomena and understanding the mechanisms underlying chromatographic behavior. This combined strategy not only discerns interaction patterns stemming from structural diversity but facilitates the rapid screening of G4s-targeting molecules.