Single-Molecule DNA Hybridization on Tetrahedral DNA Framework-Modified Surfaces

Tetrahedral DNA frameworks (TDFs) have been extensively utilized in biosensing systems. At the macroscopic level, it has been well established that TDFs, when employed as probe scaffolds, significantly enhance the interfacial DNA hybridization efficiency between target single-stranded DNA (ssDNA) and probe ssDNA at the solid–liquid interface. However, a molecular-level understanding of how TDF scaffolds facilitate this interfacial hybridization remains elusive. In this study, we employed single-molecule total internal reflection fluorescence microscopy (SM-TIRFM) to monitor the hybridization processes between freely diffusing target ssDNA and probe ssDNA with or without TDF scaffolds. Statistical analysis of interfacial hybridization kinetics at single probe sites revealed that TDF scaffolds significantly accelerate the interfacial hybridization kinetics, reducing the target dissociation time by 0.5-fold and increasing the association constant (Ka) by nearly 4-fold. This study offers new insights for the development of DNA nanostructure-based biosensors.