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

Jibin Abraham Punnoose, Kevin J. Thomas, Arun Richard Chandrasekaran, Javier Vilcapoma, Andrew Hayden, Kacey Kilpatrick, Sweta Vangaveti, Alan Chen, Thomas Banco and Ken Halvorsen ()
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Jibin Abraham Punnoose: University at Albany, State University of New York
Kevin J. Thomas: University at Albany, State University of New York
Arun Richard Chandrasekaran: University at Albany, State University of New York
Javier Vilcapoma: University at Albany, State University of New York
Andrew Hayden: University at Albany, State University of New York
Kacey Kilpatrick: University at Albany, State University of New York
Sweta Vangaveti: University at Albany, State University of New York
Alan Chen: University at Albany, State University of New York
Thomas Banco: University at Albany, State University of New York
Ken Halvorsen: University at Albany, State University of New York

Nature Communications, 2023, vol. 14, issue 1, 1-13

Abstract: Abstract 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.

Date: 2023
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DOI: 10.1038/s41467-023-36373-8

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