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.
Temperature gradient gel electrophoresis TGGE Melting temperature RNA nanoparticle pRNA 3-way junction 3WJ
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
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.
SantaLucia J Jr, Hicks D (2004) The thermodynamics of DNA structural motifs. Annu Rev Biophys Biomol Struct 33:415–440CrossRefPubMedGoogle Scholar
Chadalavada DM, Bevilacqua PC (2009) Analyzing RNA and DNA folding using temperature gradient gel electrophoresis (TGGE) with application to in vitro selections. Methods Enzymol 468:389–408CrossRefPubMedGoogle Scholar
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 41:14281–14292CrossRefPubMedGoogle Scholar
Ellington AD, Szostak JW (1990) Invitro selection of RNA molecules that bind specific ligands. Nature 346:818–822CrossRefPubMedGoogle Scholar
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 101:333–339CrossRefPubMedGoogle Scholar
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 66:359–363PubMedGoogle Scholar
Kang J, Harders J, Riesner D, Henco K (1994) TGGE in quantitative PCR of DNA and RNA. Methods Mol Biol 31:229–235PubMedGoogle Scholar
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 13:3131–3145CrossRefPubMedPubMedCentralGoogle Scholar
Danko P, Kozak A, Podhradsky D, Viglasky V (2005) Analysis of DNA intercalating drugs by TGGE. J Biochem Biophys Methods 65:89–95CrossRefPubMedGoogle Scholar
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 31:211–228PubMedGoogle Scholar
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 291:207–216PubMedGoogle Scholar
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 8:4771–4781CrossRefPubMedPubMedCentralGoogle Scholar
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 42:9996–10004CrossRefPubMedPubMedCentralGoogle Scholar
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 11:878–887CrossRefPubMedPubMedCentralGoogle Scholar
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 5:676–682CrossRefPubMedPubMedCentralGoogle Scholar