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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
SantaLucia J Jr, Hicks D (2004) The thermodynamics of DNA structural motifs. Annu Rev Biophys Biomol Struct 33:415–440
Chadalavada DM, Bevilacqua PC (2009) Analyzing RNA and DNA folding using temperature gradient gel electrophoresis (TGGE) with application to in vitro selections. Methods Enzymol 468:389–408
Nakano M, Moody EM, Liang J, Bevilacqua PC (2002) Selection for thermodynamically stable DNA tetraloops using temperature gradient gel electrophoresis reveals four motifs: d(cGNNAg), d(cGNABg),d(cCNNGg), and d(gCNNGc). Biochemistry 41:14281–14292
Ellington AD, Szostak JW (1990) Invitro selection of RNA molecules that bind specific ligands. Nature 346:818–822
Manzano M, Cocolin L, Iacumin L, Cantoni C, Comi G (2005) A PCR-TGGE (Temperature Gradient Gel Electrophoresis) technique to assess differentiation among enological Saccharomyces cerevisiae strains. Int J Food Microbiol 101:333–339
Van den Bossche A, Van Nevel C, Herman L, Decuypere J, De Smet S, Dierick N, Heyndrickx M (2001) PCR-TGGE: a method for fingerprinting the microbial flora in the small intestine of pigs. Meded Rijksuniv Gent Fak Landbouwkd Toegep Biol Wet 66:359–363
Kang J, Harders J, Riesner D, Henco K (1994) TGGE in quantitative PCR of DNA and RNA. Methods Mol Biol 31:229–235
Myers RM, Fischer SG, Lerman LS, Maniatis T (1985) Nearly all single base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis. Nucleic Acids Res 13:3131–3145
Danko P, Kozak A, Podhradsky D, Viglasky V (2005) Analysis of DNA intercalating drugs by TGGE. J Biochem Biophys Methods 65:89–95
Henco K, Harders J, Wiese U, Riesner D (1994) Temperature gradient gel electrophoresis (TGGE) for the detection of polymorphic DNA and RNA. Methods Mol Biol 31:211–228
Sorlie T, Johnsen H, Vu P, Lind GE, Lothe R, Borresen-Dale AL (2005) Mutation screening of the TP53 gene by temporal temperature gradient gel electrophoresis. Methods Mol Biol 291:207–216
Viglasky V (2013) Polyacrylamide temperature gradient gel electrophoresis. Methods Mol Biol 1054:159–171
Binzel DW, Khisamutdinov EF, Guo PX (2014) Entropy-driven one-step formation of Phi29 pRNA 3WJ from three RNA fragments. Biochemistry 53:2221–2231
Khisamutdinov EF, Jasinski DL, Guo P (2014) RNA as a boiling-resistant anionic polymer material to build robust structures with defined shape and stoichiometry. ACS Nano 8:4771–4781
Khisamutdinov EF, Li H, Jasinski DL, Chen J, Fu J, Guo P (2014) Enhancing immunomodulation on innate immunity by shape transition among RNA triangle, square and pentagon nanovehicles. Nucleic Acids Res 42:9996–10004
Severcan I, Geary C, Verzemnieks E, Chworos A, Jaeger L (2009) Square-shaped RNA particles from different RNA folds. Nano Lett 9:1270–1277
Grabow WW, Zakrevsky P, Afonin KA, Chworos A, Shapiro BA, Jaeger L (2011) Self-assembling RNA nanorings based on RNAI/II inverse kissing complexes. Nano Lett 11:878–887
Afonin KA, Bindewald E, Yaghoubian AJ, Voss N, Jacovetty E, Shapiro BA, Jaeger L (2010) In vitro assembly of cubic RNA-based scaffolds designed in silico. Nat Nanotechnol 5:676–682
Severcan I, Geary C, Chworos A, Voss N, Jacovetty E, Jaeger L (2010) A polyhedron made of tRNAs. Nat Chem 2:772–779
Binzel DW, Khisamutdinov EF, Guo PX (2014) Addition to entropy-driven one-step formation of Phi29 pRNA 3WJ from three RNA Fragments. Biochemistry 53:3709
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Benkato, K., O’Brien, B., Bui, M.N., Jasinski, D.L., Guo, P., Khisamutdinov, E.F. (2017). Evaluation of Thermal Stability of RNA Nanoparticles by Temperature Gradient Gel Electrophoresis (TGGE) in Native Condition. In: Bindewald, E., Shapiro, B. (eds) RNA Nanostructures . Methods in Molecular Biology, vol 1632. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7138-1_8
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7138-1_8
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7137-4
Online ISBN: 978-1-4939-7138-1
eBook Packages: Springer Protocols