SYBR Gold Fluorescence Quenching is a Sensitive Probe of Chitosan-microRNA Interactions
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Competitive dye displacement titration has previously been used to characterize chitosan–DNA interactions using ethidium bromide. In this work, we aim to develop a fast and reliable method using SYBR Gold as a fluorescent probe to evaluate the binding affinity between ssRNA and chitosan. The interaction of chitosan with ssRNA was investigated as a function of temperature, molecular weight and degree of acetylation of chitosan, using competitive dye displacement titrations with fluorescence quenching. Affinity constants are reported, showing the high sensitivity of the interaction to the degree of acetylation of chitosan and barely dependent on the molecular weight. We propose that the mechanism of SYBR Gold fluorescence quenching is governed by both static and dynamic quenching.
KeywordsChitosan Single-stranded RNA Dye displacement titration Fluorescence quenching
We acknowledge financial support and a PhD fellowship to BSC from German Research Council DFG (Project GRK 1549 International Research Training Group ‘Molecular and Cellular Glyco-Sciences’); from The Danish Agency for Science, Technology and Innovation, Denmark (FENAMI project 10-093456); the research leading to these results has also received funding from the European Union’s Seventh Framework Program for research, technological development and demonstration under grant agreement n° 613931. We are indebted to Prof. Susanne Fetzner (University of Münster), for her generous access to spectrophotometer and spectrofluorometer instruments. We thank Dr. Juan Pablo Fuenzalida and Dr. Kishore Babu Bobbili for their support during the experiments.
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