The Impact of ions on RNA aptamer-aminoglycoside interactions

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2019

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Fazlıoğlu, Rezzan

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Aptamers are small nucleic acid or peptide molecules that can bind to specific targets with high affinity. They have been applied for diagnostic, therapeutic and imaging purposes. In order to have the best performance of aptamers, it is worth understanding their structural dynamics. Previous studies have demonstrated that aptamer structures and thus their performances are affected by the surrounding sodium, potassium and magnesium ion concentrations. Based on these findings, we hypothesized that these ions may differently affect the binding affinity of nucleic acid aptamers. In our study, we investigated the effects of varying concentrations of sodium, potassium and magnesium ions on RNA aptamers that bind aminoglycosides. Using Isothermal Titration Calorimetry (ITC) and UV spectrophotometry, four neomycin-B binding RNA aptamers were tested in buffer solutions with different ionic contents. Our findings showed that the presence of magnesium leads to a significant change in both binding mode and affinity; whereas, potassium and sodium only affect the binding affinity of the aptamers. Depending on these results, we performed fluorescence assays with fluorophore-modified aptamers and the same buffers as used before. The change in the fluorescence signal between bound and unbound aptamers verified specific ligand binding and indicated that the concentrations and composition of these ions alter the three-dimensional structure and consequently affect the binding characteristics of aptamers. The main reason for the observed variation in aptamer performance seems to be the result of aptamers adapting to a predominant structure under a specific buffer condition. Therefore, components of the buffer systems that the aptamers are selected and optimized need to be carefully considered by researchers, because in the light of our results, the binding may get affected once the buffer system is modified by end-users.

Subject Keywords

RNA., RNA Aptamers, Metal Ions, Aminoglycosides, Isothermal Titration Calorimetry, UV Spectroscopy.

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http://etd.lib.metu.edu.tr/upload/12624760/index.pdf
https://hdl.handle.net/11511/44750

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Graduate School of Natural and Applied Sciences, Thesis

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Citation Formats

R. Fazlıoğlu, “The Impact of ions on RNA aptamer-aminoglycoside interactions,” Thesis (M.S.) -- Graduate School of Natural and Applied Sciences. Biotechnology., Middle East Technical University, 2019.