The native extracellular matrix (ECM) undergoes constant remodelling, where adhesive ligand presentation changes over time and in space to control stem cell function. As such, it is of interest to develop 2D biointerfaces able to study these complex ligand stem-cell interactions. In this study, we developed a new type of dynamic biointerface based on DNA hybridization, which can be employed to control ligand display kinetics and used to investigate how stem cells respond to dynamic ligand display. In this approach, mesoporous silica nanoparticles were functionalized with single strand DNA (MSN-ssDNA) and spin-coated on a glass substrate to create the 2D biointerface. Cell adhesive tripeptide RGD was conjugated to complementary DNA strands (csDNA) of 9, 11, or 20 nucleotides in length, to form csDNA-RGD. The resulting three csDNA-RGD conjugates could hybridize with the ssDNA on the MSN surface, presenting RGD with increased ligand dissociation rates as DNA length shortened. Our results showed that slow RGD dissociation rates led to enhanced stem cell adhesion and stem cell spreading, resulting in elongated cell morphology. Cells on surfaces with slow RGD dissociation rates also exhibited higher motility, migrating in multiple directions compared to cells on surfaces with fast RGD dissociation rates. This study contributes to the existing body of knowledge on dynamic ligand-stem cell interactions.