Single-molecule analysis of DNA base-stacking energetics using patterned DNA nanostructures

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 & PLUSMN; 0.12 kcal mol(-1) to -3.22 & PLUSMN; 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-based point accumulation in nanoscale topography (DNA-PAINT) super-resolution imaging reveals the kinetics of the free energy of base-stacking interactions of all 16 dinucleotide combinations at the single-molecule level in patterned DNA-origami nanostructures.

Automated summary

DNA-PAINT super-resolution imaging is used to measure the free energy of dinucleotide base stacking at the single-molecule level. Results show that addition of a single dinucleotide base stacking can greatly stabilize the DNA duplex nanostructure. These findings are important for designing functional DNA nanostructures and predicting the local DNA structure.