The DNA double helix structure is stabilized by base-pairing and base-stacking interactions. However, a comprehensive understanding of dinucleotide base-stacking energetics is lacking. Here we combined multiplexed DNA-based point accumulation in nanoscale topography (DNA-PAINT) imaging with designer DNA nanostructures and measured the free energy of dinucleotide base stacking at the single-molecule level. Multiplexed imaging enabled us to extract the binding kinetics of an imager strand with and without additional dinucleotide stacking interactions. The DNA-PAINT data showed that a single additional dinucleotide base stacking results in up to 250-fold stabilization for the DNA duplex nanostructure. We found that the dinucleotide base-stacking energies vary from −0.95 ± 0.12 kcal mol⁻¹ to −3.22 ± 0.04 kcal mol⁻¹ 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, and efficient assembly of higher-order DNA nanostructures. Our results will aid in designing functional DNA nanostructures, and DNA and RNA aptamers, and facilitate better predictions of the local DNA structure.

DNA 双螺旋结构通过碱基配对和碱基堆积相互作用得以稳定。然而，缺乏对二核苷酸碱基堆积能量学的全面理解。在这里，我们将纳米级形貌 (DNA-PAINT) 成像中基于多重 DNA 的点积累与设计的 DNA 纳米结构相结合，并测量了单分子水平上二核苷酸碱基堆积的自由能。多重成像使我们能够提取有或没有额外二核苷酸堆积相互作用的成像链的结合动力学。 DNA-PAINT 数据表明，单个额外的二核苷酸碱基堆积可使 DNA 双链纳米结构的稳定性提高 250 倍。我们发现，对于C|T和A|C碱基堆积，二核苷酸碱基堆积能量分别从-0.95 ± 0.12 kcal mol ^-1到-3.22 ± 0.04 kcal mol ^-1变化。我们展示了碱基堆积能量学在设计用于多重超分辨率成像的 DNA-PAINT 探针以及高阶 DNA 纳米结构的高效组装中的应用。我们的结果将有助于设计功能性 DNA 纳米结构、DNA 和 RNA 适体，并有助于更好地预测局部 DNA 结构。