JUN 25, 2015 8:00 AM PDT

Large scale purification of RNA and RNA-based Nanoparticles by Preparative Ultracentrifugation

Speakers
  • Graduate Research Assistant, University of Kentucky
    Biography
      After completion of a B.S. in Chemistry from Texas A&M University in 2008, Daniel decided to pursue his interests in research in the Department of Pharmaceutical Sciences at the University of Kentucky's College of Pharmacy. He joined the laboratory of Dr. Peixuan Guo, a world-renowned investigator in the area of bionanotechnology, in the fall of 2012. The Guo lab has pioneered the emerging field of RNA nanotechnology, even hosting a new Gordon Research Conference in 2015 dedicated to advancing this fast growing field. Daniel's research interests focus on the construction and manipulation of nanoparticles, composed mainly of RNA, publishing in several high-impact journals (ACS Nano, March 2014; Nucleic Acids Research, May 2014; ACS Nano, June 2014; Methods in Molecular Biology, March 2015). The clinical translation of RNA nanotechnology is hindered by the amounts of purified material needed for treatment. Confronted with this problem, Daniel recently spent time developing approaches for the large-scale production and purification of RNA nanoparticles, especially ultracentrifugation, which acted to retain the integrity and function of the nanostructures (Methods in Molecular Biology; May 2015).

    Abstract:
    The emerging field of RNA nanotechnology has led to the utilization of the platform in the field of bionanotechnology as sensing platforms, diverse nanoparticle construction, in vivo computing, and even in vivo drug delivery. Purification of large quantities of supramolecular RNA complexes is of paramount importance due to the large quantities of RNA needed and the purity requirements for in vitro and in vivo assays. Purification is generally carried out by liquid chromatography (HPLC), polyacrylamide gel electrophoresis (PAGE), or agarose gel electrophoresis (AGE); however, difficulties exist with each technique. Here, we describe an efficient method for the large-scale purification of RNA prepared by in vitro transcription using T7 RNA polymerase by cesium chloride (CsCl) equilibrium density gradient ultracentrifugation and the large-scale purification of RNA nanoparticles by sucrose gradient rate-zonal ultracentrifugation or cushioned sucrose gradient rate-zonal ultracentrifugation. The isolation technique has broad-reaching applicability as production of large quantities of RNA are critical to functional assays involving RNA splicing, RNA secondary structure, anti-sense RNA, and RNA:protein interaction studies.

    Learning Objectives:
    • Learn about an efficient method for the large-scale purification of RNA prepared by in vitro transcription using T7 RNA polymerase by cesium chloride (CsCl) equilibrium density gradient ultracentrifugation and the large-scale purification of RNA nanoparticles by sucrose gradient rate-zonal ultracentrifugation or cushioned sucrose gradient rate-zonal ultracentrifugation
    • Learn how this isolation technique has broad-reaching applicability as production of large quantities of RNA are critical to functional assays involving RNA splicing, RNA secondary structure, anti-sense RNA, and RNA:protein interaction studies

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