Major
Physics
Anticipated Graduation Year
2023
Access Type
Open Access
Abstract
Numerous genetic diseases arise from repetitive DNA sequences, such as CAG, that induce non-helical structures, which interfere with normal DNA processing and promote the disease state. Here, using a combination of single-molecule fluorescence microscopy and coarse-grained computational modeling, we have developed a workflow to identify the connection between the non-helical topology induced by repetitive sequences and the intrastrand interactions that specify the detected topologies. DNA three-way junctions (3WJs) were designed with two arms as helical domains and one arm consisting of a defined number of trinucleotide repeats of the sequence CAG. These DNA constructs were labeled with fluorescent dyes for single-molecule FRET experiments to measure the bend angle as a function of the number of CAG repeats. As the number of repeats increased, the DNA became increasing nonlinear and kinked. The coarse-grained modeling was performed via the oxDNA web server. With this software, thousands of potential structures were generated, which were then analyzed in terms of geometry and nucleotide-level bonding energies for intrastrand interactions. With this methodology, we are determining the size-dependent, specific nucleotide-level interactions that promote biologically relevant topologies associated with disease states.
Faculty Mentors & Instructors
Brian Cannon
Supported By
National Science Foundation
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.
Single-molecule FRET and Computational Modeling of Distinct Conformations by Repetitive DNA Junctions
Numerous genetic diseases arise from repetitive DNA sequences, such as CAG, that induce non-helical structures, which interfere with normal DNA processing and promote the disease state. Here, using a combination of single-molecule fluorescence microscopy and coarse-grained computational modeling, we have developed a workflow to identify the connection between the non-helical topology induced by repetitive sequences and the intrastrand interactions that specify the detected topologies. DNA three-way junctions (3WJs) were designed with two arms as helical domains and one arm consisting of a defined number of trinucleotide repeats of the sequence CAG. These DNA constructs were labeled with fluorescent dyes for single-molecule FRET experiments to measure the bend angle as a function of the number of CAG repeats. As the number of repeats increased, the DNA became increasing nonlinear and kinked. The coarse-grained modeling was performed via the oxDNA web server. With this software, thousands of potential structures were generated, which were then analyzed in terms of geometry and nucleotide-level bonding energies for intrastrand interactions. With this methodology, we are determining the size-dependent, specific nucleotide-level interactions that promote biologically relevant topologies associated with disease states.