Access Type

Open Access Dissertation

Date of Award

January 2025

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry

First Advisor

Christine Chow

Abstract

Nucleic acids, including DNA (deoxyribonucleic acid) and RNA (ribonucleic acids), are the fundamental building blocks of living organisms. The events that cause modification of DNA/RNA are of interest because they impact the nucleic acid function. Targeting DNA is important for treating diseases such as cancer. Cisplatin (cisPt) is a well-studied drug that targets DNA and blocks replication, but its use is limited by adverse side effects and cellular resistance. Prevoius research addressed some of these challenges through development of platinum analogues such as carboplatin and oxaliplatin. Amino acid-linked platinum analogues (AAPt) are the focus of this thesis work. AAPt complexes are synthesized using amino acids as the non-labile ligands. Previous work suggested that using positively charged amino acids such as arginine and ornithine as the ligands could have benefits such as enhanced interactions with DNA. In fact, one AAPt was shown to have good antiproliferative activity against a prostate cancer cell line with reduced activity against a normal prostate cancer cell line. These studies were the motivations to study the L-argPt and L-ornPt complexes. Moreover, the isomeric versions, D-argPt and D-ornPt, were synthesized and compared 152 with the L versions. A total of four cisPt analogues (collectively referred to as AAPt complexes) were examined in this thesis work. The antitumor properties of cisPt are related to DNA adduct formation. DNA adducts affect the structural properties of DNA, such as creating bends. Chapter 2 of the thesis, focuses on DNAs with adducts of the four AAPt complexes. The adducts were characterized by polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. AAPt complexes form DNA adducts at dGpG, dApG, dGpA, and dApA sites. Among these sites dGpA and dApA adducts were largely ignored in the past. The bend angles of DNAs containing these adducts were determined using gel mobility assays and compared with the bends created by cisPt-DNA adducts. The bend angle of a GG-containing DNA was calculated to be 30° with a cisPt adduct and 37° with AAPt (L-argPt, D-argPt, L-ornPt, and D-ornPt) adducts. The bend angles for AG-containing DNA with cisPt and AAPt adducts were similar (28° and 30°, respectively). The bend angles for GA- and AA-containing DNAs and cisPt or AAPt adducts were 29° and 22°, respectively. For both cisPt and AAPt, the DNA bend angles were adduct dependent, with decreasing bending in the following order: dGpG≥ dApG≈dGpA>dApA for cisPt and dGpG>dApG≈dGpA>dApA for AAPt DNA adducts. Reaction kinetics are an important pharmacological factor for a drug to be considered for clinical use. In Chapter 3, the reaction kinetics were examined for AAPt-DNA interactions. The studies were conducted on single-stranded DNAs containing three different adduct sites (dGpG, dApG, or dApA) and four AAPt analogues. The reaction kinetics were determined using DNA with one major adduct site and the adduct formation was monitored by gel electrophoresis. Pseudo-first order conditions were employed with excess metal complex. The reaction rates of AAPt complexes and cisPt were compared with prior studies that employed other methods such as HPLC or NMR spectroscopy. This study showed that GG-containing DNA has the highest rate of reaction with cisPt. 153 The trend for reaction rate for cisPt is GG>AA>AG DNA. The trends for the AAPt complexes varied as follows: 1) L-argPt, GG>AA≈AG DNA; 2) D-argPt, GG≈AG≈AA DNA; 3) L-ornPt, AG>AA≈GG DNA; and 4) D-ornPt, GG≈AG≈AA DNA. Canonical RNA modifications, such as 2′-O-methyl guanosine (Gm) and pseudouridine (), are involved in various roles in regulating RNA stability, transcription, and translation.145, 319 RNA oxidative damage caused by ROS is considered a significant factor in cellular dysfunction and disease.265 In Chapter 4, the roles of Gm and  in mediating oxidative stress were examined. By using radiolabeled RNA gel electrophoresis was employed to examine oxidative damage of modified RNAs and compared to unmodified RNAs. RNA sequencing techniques such as alkaline hydrolysis, chemical sequencing, and enzymatic sequencing (RNase T1) were used to map the locations of the modified sites in the RNA. The Gm-modified sites (Gm5 and Gm6) showed altered fragmentation after three different reaction times (10, 30, and 60 minutes) with the Fenton reagent which generates reactive oxygen species. Furthermore, an altered fragmentation pattern was observed at the  modification site after 30 and 60 minutes of the Fenton reaction but not for the 10-minute reaction time. These results show that the Gm and  modifications could have roles in mediating oxidative damage of RNA.

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