Using the Centrifuge Force Microscope as a Single-Molecule Technique for Studying Base-Stacking Interactions
Date of Award
Bachelor of Science
Jibin Abraham Punnoose
Pan T. X. Li
A wide range of single-molecule techniques has streamlined the process of applying and studying the effects of force on individual molecules. The centrifuge force microscope (CFM) is one such technique that allows for high-throughput single-molecule pulling experiments. It is relatively inexpensive and user-friendly, making it a suitable tool to study the effects of force on various biomolecular interactions. A benchtop centrifuge can be used to apply centrifugal force to molecular constructs that are tethered between a glass slide and a microsphere. Molecular events like bond dissociation result in the removal of the microsphere from the surface, which is observed by video microscopy during centrifugation. In this project, we used the CFM in a constant force mode to quantify the energetics of nucleotide base-stacking interactions in DNA duplexes. The interactions were quantified by measuring force-induced off-rates of DNA constructs with or without an interfacial base-stack. Differences were observed in the off-rates between the constructs containing additional base-stacks at the interface relative to those without, and this difference was used to calculate the energy contribution of each base-stack. A global trend was identified, with purine-purine base-stacks being the most stable, then purine-pyrimidine base-stacks, and finally, pyrimidine-pyrimidine base-stacks being the least stable. This data will be useful in improving molecular modeling and in designing more stable DNA nanostructures. Similar experimental designs can be used to study other biomolecular interactions like receptor-ligand binding or enzyme activity assays. Moreover, this spotlights the capabilities of the CFM as a versatile singlemolecule technique.
Thomas, Kevin, "Using the Centrifuge Force Microscope as a Single-Molecule Technique for Studying Base-Stacking Interactions" (2022). Biological Sciences. 82.