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

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.

Versatile DNA Balances via Adjacent Base Stacking for Homogeneous Assay of Energy Parameters, Small Molecules, And Ribonuclease

“…Quantifying the energy contribution of base stacking has been of considerable interest via several methods, such as thermal denaturation methods, , electrophoretic analysis of equilibrium, and single-molecule approaches. − However, the methods often require complicated operations and it is difficult to quantify the energy parameter of base stacking in a native environment. Similar to a real balance, here, changes in thermodynamic parameters of the DNA balance can be employed to calculate the energy contribution of base stacking.…”

Assessing the Current State of Amber Force Field Modifications for DNA─2023 Edition

“…This observation could not be the case for other systems, and research about the influence of salt models and salt concentration on nucleic acids in MD simulations is ongoing . On the topic of base pair stacking, several groups have performed studies with seemingly contradicting resultssome have seen parm99 parameters result in overstacking and some observed understacking . The overstacking present, though notably mild, requires more work to be done to investigate the potential sequence dependence of base pair stacking in double-stranded nucleic acid systems.…”

“…The overstacking present, though notably mild, requires more work to be done to investigate the potential sequence dependence of base pair stacking in double-stranded nucleic acid systems. Experimentally accurate description of base pair stacking is an ever-present issue in all the force fields evaluated hereparm99-based force fields with varying results − and Tumuc1 overstabilizing base pair stacking compared to the experiment. , Possible solutions may lie in the refinement of glycosidic terms or adjustments in partial charge distributions, but further investigation is needed to address this characteristic that is critical to nucleic acid helical stability.…”

van der Waals Parameter Scanning with Amber Nucleic Acid Force Fields: Revisiting Means to Better Capture the RNA/DNA Structure through MD

“…Decades of benchmarking and evaluations have provided a thorough record of the current limitations of the available Amber nucleic acid force fields. ,− These works have uncovered a hyper-tendency for hydrogen bonding, overstacking, and the overstabilization of infinite helices, to name a few. ,, Many of these issues have been attributed to a misbalance in the van der Waals (vdW) parameters, which have not been improved since their development in tandem with the original parm94 force field . At their conception, Amber vdW parameters were defined by atom typefor example, all sp3-hybridized carbon atoms share the same vdW parameters, as do all sp2-hybridized carbon atoms. , These initial carbon and hydrogen atom type vdW parameters were originally fit to reproduce the densities and enthalpies of vaporization for alkanes and benzenes. , Concurrently, oxygen, nitrogen, and phosphorus atom vdW parameters were developed to reproduce neat liquid properties, as well as to fit lattice energies and crystal structures .…”