Date of Award

Spring 5-2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry and Biochemistry

Committee Chair

Dr. Faqing Huang

Committee Chair Department

Chemistry and Biochemistry

Committee Member 2

Dr. Sabine Heinhorst

Committee Member 2 Department

Chemistry and Biochemistry

Committee Member 3

Dr. Vijayaraghavan Rangachari

Committee Member 3 Department

Chemistry and Biochemistry

Committee Member 4

Dr. YanLin Guo

Committee Member 4 Department

Biological Sciences

Committee Member 5

Dr. Douglas Masterson

Committee Member 5 Department

Chemistry and Biochemistry

Abstract

Coenzyme A is an indispensable molecule in all known life with roles in metabolism, gene regulation, and macromolecule synthesis. As CoA is derived from RNA itself, it’s incorporation into RNA by in vitro methods has proven useful in research probing the origin of life based on the RNA World theory. The discovery in contemporary bacteria of RNA modified with CoA, however, provided an unexpected twist to previously well-characterized bacterial systems. The identity of sequences associated with CoA-RNA has been elusive since their discovery in 2009 based on the difficulties in isolation while maintaining RNA quality. The aim of this study is to develop a specific CoA-RNA capture protocol to uncover the sequence identities for the further understanding of CoA-RNA function. While the free thiol of CoA can be used to purify the CoA-RNA from total bacterial RNA, the success of process depends on the specific release from the purification matrix. This work details the characterization of recombinant Nudix CoA pyrophosphohydrolases from various organisms for their use in CoA hydrolysis and CoA-RNA metabolism, as well as for the development and successful implementation of a specific CoA-RNA capture protocol. The use of this protocol for the construction of high quality NGS libraries is vital to the elucidation of CoA-RNA function via bioinformatic analysis, for its potential ranges from a simple stabilization adduct to contemporary examples of RNA World era coenzyme-conjugated ribozymes.

ORCID ID

orcid.org/0000-0002-0673-1459

Available for download on Wednesday, May 16, 2018

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