Date of Completion
Spring 5-1-2025
Thesis Advisor(s)
Yongku Cho
Honors Major
Chemical Engineering
Disciplines
Biochemical and Biomolecular Engineering
Abstract
Engineered RNA-binding proteins (RBPs) show promise for diverse applications in research and therapeutic development. Modulating the interaction between YTH-domain containing proteins and methylated RNA ligands could result in a high affinity, highly specific binding protein for the advancement of research tools in neurodegenerative disease. N6-methyladenosine (m6A) modified RNA contributes to stress granule formation that promotes neurotoxicity in neurodegenerative disease, with concentrations of m6A-RNA increased fivefold in Alzheimer’s Disease. Further affinity maturation of the YTH-m6A interaction through directed evolution of the YTH domain could produce a novel protein with increased affinity better suited for the development of biological detection tools or therapeutic proteins. Here, I explore the design of a yeast surface display platform for the engineering of RBPs to m6A-RNA ligands. Furthermore, I explored the efficacy of Nanopore sequencing to characterize mutant libraries for subsequent directed evolution experiments. While there appears to be evidence that RNA-RBP interactions can be observed with this platform, further experiments with more robust expression labeling are necessary to proceed with the directed evolution of the YTH protein using yeast surface display. Additionally, while concatemer formation during sequencing library preparation compromised the reliability of high-throughput DNA sequencing results, there is still promise for this approach as well in future experiments.
Recommended Citation
Horvath, Katelynn E., "Engineering Affinity of YTH Protein to M6A-RNA" (2025). Honors Scholar Theses. 1093.
https://digitalcommons.lib.uconn.edu/srhonors_theses/1093
