Proximity‐Based Super‐Resolution Imaging Enabled by DNA Base‐Stacking Interactions

Abstract Super‐resolution imaging has gained significant traction in recent years due to its unprecedented ability to visualize target biomolecules at nanometer resolution. Here, the capability is demonstrated to detect target pairs that are in close proximity by exploiting base‐stacking interactions between two DNA strands, each labelling one target. The DNA probes hybridize transiently with the each other only when both target molecules are proximal, thus creating a hybridization site for fluorophore‐conjugated DNA strand, called imager. In this design, hybridization and co‐axial base‐stacking act synergistically to enable imager binding, with stacking interactions providing essential stabilization that allows for transient hybridization. This synergy generates the stochastic binding events required for DNA‐PAINT imaging, which it call Stack‐Proximity‐PAINT (Stack‐pPAINT). To gain mechanistic insights into hybridization of the probe, atomistic equilibrium and steered molecular dynamics (MD) simulations are performed. The simulations reveal that DNA base‐stacking and fluorophore stacking together stabilize the imager. Programmable DNA nanostructures are utilized to benchmark the applicability of Stack‐pPAINT. As a cellular proof of concept, microtubular structures are visualized using Stack‐pPAINT with antibodies targeting both alpha‐ and beta‐tubulin molecules. This probe technology offers promising applications in cell biology research aimed at elucidating spatial interactomes at high resolution within cells.