Engineering quantum dot surfaces to preserve protein-DNA interactions for single-molecule visualization

Quantum dots (QDs) are fluorescent nanoparticles widely used for single-molecule imaging because of their exceptional brightness and photostability. However, the impact of QD surface chemistry on biomolecular interactions has not been systematically investigated. Here, we report that commercial QDs unexpectedly destabilize protein-DNA complexes by inducing protein dissociation from DNA. Using the human nucleotide excision repair protein, xeroderma pigmentosum complementation group A (XPA) as a model system, we demonstrate that antibody-conjugated QDs promote dissociation of XPA from DNA substrates, independently of sizes and surface modification of QDs, antibody types, epitope tags, buffer conditions, or DNA structures. We find that polyethylene glycol (PEG), a common polymer coating on QD surfaces, is the primary factor responsible for this effect. To tackle this problem, we engineered QDs with precisely controlled surface polymer compositions. By systematically changing the ratio of anchoring, hydrophilic, and PEG-based functional groups, we find that reducing PEG density below a critical threshold effectively suppresses protein dissociation while maintaining excellent colloidal stability and brightness. Furthermore, antibodies conjugated via click chemistry between azide groups and DBCO enabled specific labeling of XPA without perturbing the DNA binding activity. Using these optimized QDs, we conducted single-molecule DNA curtain assays to visualize XPA-DNA interactions. QD-labeled XPA exhibits one-dimensional diffusion with frequent pausing on undamaged DNA. DNA curtain assays revealed that XPA preferentially binds DNA bubbles and searches for bubble structures through both one-dimensional diffusion and three-dimensional collision. Quantitative analysis showed that three-dimensional collision is the dominant pathway for bubble recognition. Taken together, our results uncover a previously unrecognized limitation of PEG-coated QDs in single-molecule studies and provide an improved surface-engineering strategy to preserve native protein-DNA interactions. Newly engineered QDs establish robust platforms for accurate single-molecule visualization of biomolecular processes.