Tag: Victoria DeRose

New Pt(II) Complexes for the Investigation of Copper-Mediated Degradation in Pt-Bound RNA Click Reactions

Presenter: Lindsay Guzman

Mentor: Victoria DeRose

Oral Presentation

Major: Chemistry

RNAs contribute to a wide range of essential biological processes such as protein function, catalysis, transcriptional and translational regulation. Small-molecule binders, such as the platinum(II) anticancer drug cisplatin, can be used to probe cellular RNA structures and functions. We are focusing on the functionalization of Pt complexes with azide and alkyne moieties that may allow for the subsequent purification and high-throughput sequencing of Pt-RNA adducts in the copper-catalyzed Huisgen cycloaddition (click) reaction. The click reaction involves the formation of a thermodynamically stable five-membered ring between an azide and alkyne in the presence of a Cu catalyst. Because of the reactive nature of the necessary Cu catalyst, it is speculated that Cu is facilitating observed RNA degradation in model reactions, thus lowering the efficiency and usefulness of the post-treatment click modifications. Three new Pt(II) complexes that vary in linker length from the platinum center to the copper-catalyzed click reaction site will be synthesized to probe the possible influence of click-mediated Cu recruitment on cleavage of the oligonucleotide, which will be determined by gel chromatography. This will allow for further investigation of additional and undesired copper-mediated reactivity as well as improved yield of Pt-bound RNA click reactions, which will help elucidate the biological processes of RNA.

Development of a Pull-down Procedure for Isolating Platinated Cellular Molecules

Presenter: Anna Hickey

Faculty Mentor: Victoria DeRose, Geri Richmond

Presentation Type: Poster 69

Primary Research Area: Science

Major: Biochemistry

Funding Source: Presidential Undergraduate Research Scholars Program, University of Oregon, $5,000.00 research stipend; Scholarships for Oregon Scientists, University of Oregon and National Science Foundation, $2,000.00 research stipend

Cisplatin is a commonly used anti-cancer therapeutic; however, its mechanism of inducing cell death is not well understood. In order to identify and isolate cisplatin’s cellular targets for characterization, our lab utilizes the
“click” reaction (a physiologically stable and high yielding reaction that produces no harmful byproducts) to attach fluorescent compounds or other small molecules to platinated cellular targets such as DNA, RNA, and proteins. In this project, I optimized an in vitro pull-down procedure using streptavidin-coated magnetic beads to separate platinated cellular targets from unplatinated molecules. I first treated target DNA with a click-functionalized platinum reagent, then clicked that compound to a double-stranded DNA linker. The opposite end of this linker contains a biotin molecule, which interacts strongly with the streptavidin-coated magnetic beads through the streptavidin-biotin interaction. Using a powerful magnet, I separated platinated and clicked DNA attached to the beads from unreacted DNA, then confirmed the desired species of DNA was pulled down using polyacrylamide gel electrophoresis (PAGE), a method by which DNA or proteins can be separated by size. I determined that increasing the incubation time of the beads with the platinated DNA increased elution yields. Furthermore, elution temperatures above 90° C also increase the elution yield. Optimizing this pull-down technology will allow us to better characterize platinated molecules, and will ultimately improve our understanding of cisplatin’s cell-death inducing mechanisms.