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

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Base stacking interactions are crucial for DNA/RNA structure and function.
• Previous studies estimated pairwise stacking energies, but individual base contributions were unknown.

Purpose of the Study:

• To quantify individual base stacking energies using high-throughput single-molecule experiments.
• To investigate the impact of modified nucleotides and fluorophores on stacking interactions.
• To demonstrate the influence of base stacking on DNA nanostructure stability, enzymatic ligation, and molecular dynamics force fields.

Main Methods:

• Utilized Centrifuge Force Microscopy for high-throughput single-molecule measurements.
• Experimentally determined stacking energies between adjacent DNA bases.
• Assessed effects of nucleotide modifications (phosphorylation, methylation, RNA) and fluorophore attachment.

Main Results:

• Identified strongest stacking energies between purines (G|A: -2.3 ± 0.2 kcal/mol) and weakest between pyrimidines (C|T: -0.5 ± 0.1 kcal/mol).
• Found no significant effect of phosphorylated, methylated, or RNA nucleotides on stacking energy.
• Observed a reduction in stacking energy due to fluorophore modification.

Conclusions:

• Base stacking energies vary significantly between different base pairs.
• Base stacking influences DNA nanostructure stability, enzymatic ligation kinetics, and molecular dynamics simulations.
• Provides fundamental insights into DNA interactions for biological understanding and biotechnological applications.