High-throughput single-molecule quantification of individual base stacking energies in nucleic acids

Base stacking interactions between adjacent bases in DNA and RNA are known to be important for many biological processes, for drug development, and in other biotechnology applications. While previous work has estimated base stacking energies between pairs of bases, the individual contributions of each base to the stacking interaction has remained unknown. Here, we developed a novel methodology using a custom Centrifuge Force Microscope to perform high-throughput single molecule experiments to measure base stacking energies between individual adjacent bases. We found stacking energies strongest between purines (G|A at -2.3 +/- 0.2 kcal/mol) and weakest between pyrimidines (C|T at -0.4 +/- 0.1 kcal/mol). Hybrid stacking with phosphorylated, methylated, and RNA bases had no measurable effect, but a fluorophore modification reduced stacking energy. The implications of the work are demonstrated with three applications. We experimentally show that base stacking design can influence assembly and stability of a DNA nanostructure, modulate kinetics of enzymatic ligation, and determine accuracy of force fields in molecular dynamics (MD) simulations. Our results provide new insights into fundamental DNA interactions that are critical in biology and can inform rational design in diverse biotechnology applications.