DNA double helix structure is stabilized by the base-pairing and the base-stacking interactions. Base-stacking interactions originating from hydrophobic interactions between the nucleobases predominantly contribute to the duplex stability. A comprehensive understanding of dinucleotide base-stacking interactions is lacking owing to the unavailability of sensitive techniques that can measure these weak interactions. Earlier studies attempting to address this question only managed to estimate the base-pair stacking interactions, however, disentangling individual base-stacking interactions was enigmatic. By combining multiplexed DNA-PAINT imaging with designer DNA nanostructures, we experimentally measure the free energy of dinucleotide base-stacking at the single-molecule level. Multiplexed imaging enabled us to extract binding kinetics of an imager strand with and without additional dinucleotide stacking interactions in a single imaging experiment, abolishing any effects of experimental variations. The DNA-PAINT data showed that a single additional dinucleotide base-stacking results in as much as 250-fold stabilization of the imager strand binding. We found that the dinucleotide base-stacking energies vary from -1.18 ± 0.17 kcal/mol to -3.57 ± 0.08 kcal/mol for C|T and A|C base-stackings, respectively. We demonstrate the application of base-stacking energetics in designing DNA-PAINT probes for multiplexed super-resolution imaging. Our results will aid in designing functional DNA nanostructures, DNA and RNA aptamers, and facilitate better predictions of the local DNA structure.

Super-resolution imaging is becoming a commonly employed tool to visualize biological targets in unprecedented detail. DNA-PAINT is one of the single-molecule localization microscopy-based super-resolution imaging modalities allowing for ultra-high-resolution imaging...