Honors Program
Document Type
Thesis
Date of this Version
2022
Citation
Brady, S. 2022. Optimization and Standardization of DMS Chemical Mapping Experiments for Probing RNA 3D Structure. Undergraduate Honors Thesis. University of Nebraska-Lincoln.
Abstract
RNA plays a significant and crucial role in biological processes. The properties of RNA are ultimately responsible for each molecule’s unique function. The configuration and structural integrity of RNA is important for its functionality. Chemical mapping detailed in this thesis can be used to analyze the 3D structure of an RNA sequence. RNA is modified via methylation of adenine and cytosine bases using dimethyl sulfate, then reverse transcribed to DNA, then sequenced. The sites where the bases were methylated become mutations in the DNA sequence, allowing for identification of these locations when analyzing sequencing data. Conditions for modification can be altered to provide insight into the structure of the compound in vitro. This thesis details optimizations and changes made to chemical mapping protocols to increase data accuracy, experiment reproducibility, and efficiency. The major changes made include altering of modification time and amount of dimethyl sulfate (DMS) used, increasing magnesium amounts added to the reaction to aid in folding, and altering of purification methods and DNA processing.
Comments
Copyright Sarah Brady 2022.