Poster abstracts
Poster number 97 submitted by Daniel Dayeh
Quadruplex-mediated affinity purification of DNA-induced slicing complex
Daniel M. Dayeh (The Ohio State University), Elaina P. Boyle (The Ohio State University), Besik Kankia (The Ohio State University), Karin Musier-Forsyth (The Ohio State University), Kotaro Nakanishi (The Ohio State University)
Abstract:
Budding yeast Argonaute, a central enzyme in the RNA interference pathway, was recently loaded with a DNA guide and shown to assemble a functional DNA-induced slicing complex (DISC) that can cleave highly-structured RNAs in vitro. A major limitation in the preparation of recombinant eukaryotic Argonaute is the difficulty of removing host-derived endogenous cellular RNAs that remain tightly bound to the majority of the population, leaving only small amounts of protein available for loading with synthetic RNA or DNA guides. This drawback has hampered the development of more advanced Argonaute-mediated applications. Here, we use the recently-described quadruplex-and-Mg2+ connection technology to develop a nucleic acid-based affinity tag for the isolation of homogeneous DISCs. This method allows active and desirable DISCs to be purified away from contaminating RNA-bound complexes that limit the current use of DISC. The simple, cost-effective method presented here increases the potential applications of Argonaute-based tools in biotechnology and therapeutics.
References:
Dayeh, D.M., Cantara, W.A., Kitzrow, J.P., Musier-Forsyth, K., Nakanishi, K. . (2018) Argonaute-based programmable RNase as a tool for cleavage of highly-structured RNA Nucleic Acids Res.
Keywords: RNAi, DISC , programmable RNase