Surface Plasmon Resonance: A Useful Strategy for the Identification of Small Molecule Argonaute 2 Protein Binders

Poser E, et al., 1517:223-237, Methods Mol Biol., 2017

The Argonaute proteins play a key role in RNA silencing processes. Small non-coding RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs) guide Argonaute proteins such as Argonaute 2 protein (AGO2) to their specific targets through base pairing resulting in silencing due to mRNA cleavage. Described herein is the use of Surface Plasmon Resonance (SPR) methods to characterize the binding interaction between Argonaute 2 and the small molecule, BCI-137. A Pall ForteBio Pioneer system equipped with COOH5 sensor chip was used to perform all SPR experiments. AGO2 was immobilized onto sensor chip surface using standard amine coupling chemistry (NHS/EDC). Any residual active sites were blocked using ethanolamine. FastStep injections of BCI-137 were performed using seven serial doubling dilutions. Sucrose was used as a bulk reference standard and internal control for the experiment. Data obtained were globally fitted using a simple 1:1 Langmuir model. The equilibrium dissociation constant was determined using a scatchard analysis of the dependence of Req on the concentration of analyte concentration. Overall results of this study demonstrate how SPR technology can be used to monitor biomolecular interactions in real time.

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