Date of Award

Summer 8-2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry and Biochemistry

Committee Chair

Dr. Anthony Bell

Committee Chair Department

Chemistry and Biochemistry

Committee Member 2

Dr. Gordon Cannon

Committee Member 2 Department

Chemistry and Biochemistry

Committee Member 3

Dr. Vijay Rangachari

Committee Member 3 Department

Chemistry and Biochemistry

Committee Member 4

Dr. Karl Wallace

Committee Member 4 Department

Chemistry and Biochemistry

Committee Member 5

Dr. Philip Bates

Committee Member 5 Department

Chemistry and Biochemistry

Abstract

The studies presented in section 1 (Chapters I-IV) focus on the design and development of nucleic acid four-way junctions (4WJs) to target a member of the high mobility group (HMG) proteins, the proinflammatory cytokine high mobility group box 1 protein (HMGB1). In the present study, hybrid PNA-DNA 4WJs based on a model DNA 4WJ were constructed to improve the thermal stability of 4WJs while maintaining strong binding affinity toward HMGB1. An electrophoretic mobility shift assay (EMSA) was used to examine the binding affinity of an isolated DNA binding domain of HMGB1, the HMGB1 b-box (HMGB1b), toward a set of PNA-DNA hybrid 4WJs. EMSAs showed that HMGB1b recognizes single-PNA hybrid 4WJs with a similar affinity to the DNA control. Circular dichroism (CD) spectroscopy was used to examine the structure and monitor thermal transitions of hybrid PNA-DNA four-way junctions in low (0.01mM Mg2+) and high (2.00mM Mg2+) ionic strength environments. CD analysis suggests a large deviation in helical structure between DNA and PNA hybrid junctions. Blunt-ended hybrid junctions b4WJ-PNA1 and b4WJ-PNA3 had higher melting temperatures (Tms) than their full-length counter parts with DTms of 1.55 and 5.43°C, respectively. Junction b4WJ-PNA3 was shown capable of binding HMGB1b with an affinity similar to that of its parent DNA junction and has a Tm of 41.18°C, 1.14°C higher than its parent DNA junction J1 and well above normal body temperature, suggesting that b4WJ-PNA3 may be a viable therapeutic agent for targeting HMGB1 in vivo.

In the study presented in section 2 (chapters 5-8), the amino acid (AA) selectivity of aminoacyl-tRNA synthetases (aaRSs) were examined to investigate a possible role for aaRSs in genetic code development. A radiometric assay was used to perform an exhaustive survey of the 20 natural AAs vs. the 20 aaRSs from Escherichia coli (E. coli). This study presents an AA chronology of Early vs. Late AAs based on misactivation frequency. Findings were compared with current theories on the evolution of AA recruitment and codification, and show a correlation between misactivation frequency and the order of AA codification, suggesting that aaRSs may have played a role in the process of AA codification.

ORCID ID

orcid.org/0000-0001-8492-8399

Previous Versions

Jul 27 2016

Available for download on Sunday, August 05, 2018

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