Toward the identification of anionic guanines in vivo
Open Access
- Author:
- Duncan, Glenn
- Millennium Scholars Program:
- Biology (BIOL)
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisor:
- Philip C Bevilacqua, Thesis Supervisor
Georjanne Phyllette Williams Rosa, Advisor - Keywords:
- Anionic guanines
in vivo - Abstract:
- The charge state of RNA nucleobases can impact RNA folding, which in turn may affect gene regulation. 1-Ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) and glyoxal have been developed as chemical reagents that react with and covalently modify guanine nucleobases in vivo. EDC tends to react with uncharged guanines found in the neutral intracellular environment, whereas glyoxal reacts with anionic guanines that are not typically found in the intracellular environment. Previous in vivo treatment of rice (Oryza sativa) seedlings with glyoxal and EDC have identified potential anionic guanines. The experiments described herein were designed to use a cost-efficient approach of finding more anionic guanine candidates in vivo. The first step was to determine the efficacy of EDC reactivity in vitro using RNA extracted from rice seedlings. Upon finding that EDC selectively modified uridines and guanines in vitro, the next steps were to treat rice seedlings with EDC, determine whether EDC modified RNA within intact cells, and pull down RNA targets. Chapter 1 of this thesis introduces RNA structure and the use of probing reagents to elucidate structure. Next, chapter 2 covers crystal structure analysis conducted in this research to examine the environment surrounding these guanines, and several interactions that were identified that could promote an anionic charge. Chapter 3 discusses the observation of EDC modification with uridines and guanines in vitro. This result allowed further in vivo treatment of rice seedlings with EDC, the extracted RNA from which would be used for all subsequent experiments. Lastly, Chapter 4 mentions experiments that have yet to be performed and future directions. Specifically, probe design methodology to pull down selected target RNAs, pulling down the RNAs of interest, isolating RNAs, and preparing them into cDNA libraries for sequencing. The final goal would be to analyze sequencing data to indicate the presence and location of anionic guanines.