Thinking inside the box: designing, implementing, and interpreting thermodynamic cycles to dissect cooperativity in RNA and DNA folding.
Double and triple mutant thermodynamic cycles provide a means to dissect the cooperativity of RNA and DNA folding at both the secondary and tertiary structural levels through use of the thermodynamic box or cube. In this article, we describe three steps for applying thermodynamic cycles to nucleic acid folding, with considerations of both conceptual and experimental features. The first step is design of an appropriate system and development of hypotheses regarding which residues might interact. Next is implementing this design in terms of a tractable experimental strategy, with an emphasis on UV melting. The final step, and the one we emphasize the most, is interpreting mutant cycles in terms of coupling between specific residues in the RNA or DNA. Coupling free energy in the absence and presence of changes elsewhere in the molecule is discussed in terms of specific folding models, including stepwise folding and concerted changes. Last, we provide a practical section on the use of commercially available software (KaleidaGraph) to fit melting data, along with a consideration of error propagation. Along the way, specific examples are chosen from the literature to illustrate the methods. This article is intended to be accessible to the biochemist or biologist without extensive thermodynamics background.
Siegfried
Nathan A
NA
Department of Chemistry, Pennsylvania State University, University Park, Pennsylvania, USA.
Bevilacqua
Philip C
PC
D000818:Animals; D004247:DNA / Q000032:analysis* / Q000737:chemistry*; D009690:Nucleic Acid Conformation / Q000528:radiation effects; D009691:Nucleic Acid Denaturation; D012313:RNA / Q000032:analysis* / Q000737:chemistry*; D013816:Thermodynamics
D012313:RNA; D004247:DNA