Base stacking interactions between adjacent bases in DNA and RNA are important for many biological processes and in biotechnology applications. Previous work has estimated stacking energies between pairs of bases, but contributions of individual bases has remained unknown. Here, we use a Centrifuge Force Microscope for high-throughput single molecule experiments to measure stacking energies between 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 −0.5 ± 0.1 kcal/mol). Hybrid stacking with phosphorylated, methylated, and RNA nucleotides had no measurable effect, but a fluorophore modification reduced stacking energy. We experimentally show that base stacking can influence stability of a DNA nanostructure, modulate kinetics of enzymatic ligation, and assess accuracy of force fields in molecular dynamics simulations. Our results provide insights into fundamental DNA interactions that are critical in biology and can inform design in biotechnology applications.

DNA is stabilized by inter-strand base pairing and intra-strand base stacking. Untangling these energy contributions is challenging, but has implications for understanding biological processing of DNA, and for many aspects of biotechnology including drug discovery, molecular modeling and DNA nanotechnology. Here, we developed novel DNA constructs and performed single molecule experiments using a custom Centrifuge Force Microscope (CFM) to probe energetics of base stacking interactions between single bases. Collecting rupture statistics from over 30,000 single-molecule tethers, we quantified 10 unique base stacking combinations and 4 modified nucleotides. For canonical bases, we found stacking energies strongest for purines (G|A at -2.3 +/- 0.2 kcal/mol) and weakest for pyrimidines (C|T at 0.4 +/- 0.1 kcal/mol). Among hybrid stacking with modified nucleotides, only a bulky fluorophore modification reduced stacking energy while phosphorylated, methylated, and RNA bases had little effect. We demonstrate the implications of the work with two biotechnology applications: using interfacial base stacks to tune the stability of a DNA tetrahedron, and to alter the kinetics of enzymatic ligation. These results provide new insights into fundamental DNA interactions that are critical in biology and biotechnology.

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