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Evaluation of Thermal Stability of RNA Nanoparticles by Temperature Gradient Gel Electrophoresis (TGGE) in Native Condition

  • Kheiria Benkato
  • Benjamin O’Brien
  • My N. Bui
  • Daniel L. Jasinski
  • Peixuan Guo
  • Emil F. KhisamutdinovEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1632)

Abstract

Temperature gradient gel electrophoresis (TGGE) is a powerful tool used to analyze the thermal stabilities of nucleic acids. While TGGE is a decades-old technique, it has recently gained favor in the field of RNA nanotechnology, notably in assessing the thermal stabilities of RNA nanoparticles (NPs). With TGGE, an electrical current and a linear temperature gradient are applied simultaneously to NP-loaded polyacrylamide gel, separating the negatively charged NPs based on their thermal behavior (a more stable RNA complex will remain intact through higher temperature ranges). The linear temperature gradient can be set either perpendicular or parallel to the electrical current, as either will make the NPs undergo a transition from native to denatured conformations. Often, the melting transition is influenced by sequence variations, secondary/tertiary structures, concentrations, and external factors such as the presence of a denaturing agent (e.g., urea), the presence of monovalent or divalent metal ions, and the pH of the solvent. In this chapter, we describe the experimental setup and the analysis of the thermal stability of RNA NPs in native conditions using a modified version of a commercially available TGGE system.

Key words

Temperature gradient gel electrophoresis TGGE Melting temperature RNA nanoparticle pRNA 3-way junction 3WJ 

Notes

Acknowledgment

We thank Seth Abels for proofreading this work and leaving valuable comments. The research was supported by Department of Chemistry BSU start-up funds, Chemistry Research Immersion Summer Program (CRISP) at BSU and Indiana Academy of Science grant # G9000602A to Emil Khisamutdinov.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Kheiria Benkato
    • 1
  • Benjamin O’Brien
    • 1
  • My N. Bui
    • 1
  • Daniel L. Jasinski
    • 2
    • 3
    • 4
  • Peixuan Guo
    • 2
  • Emil F. Khisamutdinov
    • 1
    Email author
  1. 1.Department of ChemistryBall State UniversityMuncieUSA
  2. 2.Department of Physiology and Cell Biology, College of PharmacyThe Ohio State UniversityColumbusUSA
  3. 3.Division of Pharmaceutics and Pharmaceutical Chemistry, College of PharmacyThe Ohio State UniversityColumbusUSA
  4. 4.Dorothy M. Davis Heart and Lung Research InstituteThe Ohio State UniversityColumbusUSA

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