Experimentally verified flexible molecular docking and dynamic simulation of aptamer with intracellular proteins...

Despite the rising spotlight on biotargeting aptamers, their mechanism of regulating cellular functions remained elusive due to lack of systematic method to investigate their intracellular behavior. This study systematically established a complete workflow including DNA secondary and 3D structure prediction, flexibilization, docking, experimental validation, and molecular dynamic (MD) simulation. RNAfold was demonstrated to provide more accurate ssDNA secondary structure predictions and compatibility for flexible docking. Feasibility of a novel direct prediction tool of DNA 3D structure, 3dDNA, has first been proven with similar reliability and better data stability in flexible docking compared to indirect prediction by RNAComposer. Flexible docking by AutoDock Vina exhibited higher reliability, while rigid docking was less reliable. Docking results were influenced by secondary and 3D structures, but the proteins' inherent affinity to nucleic acids was the key determinant. Aptamer bound to proteins with non-specificity (K_D > 100 nM) and affinities (R_max) exponentially correlated to flexible docking scores, necessitating further MD validation and identification of binding sites. Via the established workflow, binding sites of stem cell-recruiting aptamer Apt-19s on its known target (ALPL) was identified, Sec24B was first screened as its potential intracellular targets, providing theoretical guidance and feasible methodology for future exploration of aptamer biotargeting mechanisms.