Open Access Dissertation
Date of Award
Christine S. Chow
RNAs are crucial for many cellular functions. Thus, studying ligand-RNA interactions and their dynamics in response to changes in the surrounding environment is important. In spite of the well-known DNA coordination, current research also indicates cisplatin binding to RNA. Kinetic studies of rRNA platination reactions are largely unexplored. This research was conducted to achieve two objectives. First, a broad kinetic study was carried out to investigate the cisplatin-rRNA interactions. The structure, function, and ligand interactions depend on RNA microenvironments. Second, the application of platination kinetics as a tool to interrogate RNA electrostatic environments was explored.
Three model rRNA hairpins from E. coli ribosome were selected. Two helix 69 (H69) constructs, modified H69 (with pseudouridine) and unmodified H69 (without pseudouridine), and the 790 loop, which has an identical size and nucleotide composition to unmodified H69, were used. Prior to kinetic studies, cisplatin targets on each RNA were determined using RNase T1 mapping combined with MALDI MS, and dimethyl sulfate (DMS) probing. The kinetic studies were carried out under pseudo-first-order conditions and electrostatic properties were evaluated using Brønsted-Debye-Hückel and polyelectrolyte theories.
RNase T1 mapping with MALDI MS and dimethyl sulfate (DMS) probing revealed GpG sites as cisplatin targets on RNA. The DMS probing further revealed platination-induced structural changes in RNA. Both the RNA sequence and modified nucleotides showed an impact on platination rates. Kinetic data showed that the platination rate is dependent on cations and the abundance of active cisplatin complexes. Structure, pseudouridylation, availability of active cisplatin species, and cation/Pt+ electrostatic competitions all impact platination of the two H69 RNAs. Probing neomycin-H69 interactions by platination kinetics indicated that structural changes in modified H69 upon aminoglycoside binding could also impact the platination kinetics. Electrostatic models revealed that nucleotide sequence, cations, and H+ ions impact the global RNA electrostatics. The similar global electrostatic properties between the two H69 RNAs indicated that structure-dependent electrostatic changes in modified H69 could be limited to the loop region.
In conclusion, this thesis work showed that both intrinsic RNA characteristics such as structure, sequence, and dynamics, as well as bulk solution conditions (e.g. cations and pH), impact cisplatin-RNA interactions. The RNA electrostatic parameters determined in this thesis work illustrated platination kinetics can be used as an informatory tool for probing dynamic RNA microenvironments.
Dedduwa-Mudalige, Gayani, "Platination Kinetics: Insight Into Rna-Cisplatin Interactions As A Probe For Rna Microenvironments" (2015). Wayne State University Dissertations. 1332.