Analyzing and predicting the thermodynamic effects of the metabolite trehalose on nucleic acids.
There is a lot of interest in exactly how nucleic acid duplexes are affected by the addition of certain stabilizing and destabilizing metabolites. Unfortunately, the differences in reaction conditions between published reports often precludes a comparison of the results, effectively preventing a cohesive strategy for predicting additive effects on nucleic acid stability. This information is critically important for obtaining a fundamental understanding of how additives, including metabolites, alter DNA and RNA stability and structure. We now show that the destabilization of nucleic acids by the metabolite trehalose in standard optical melting buffer (20 mM sodium cacodylate, 1M NaCl, and 0.5 mM EDTA) differs from that of a common PCR buffer, and a simulated physiological buffer, with up to an 8°C melting temperature difference. We also demonstrate that the extent of DNA destabilization due to trehalose depends on DNA length and depends on percent GC content, at least for the primer-length duplexes studied here. Furthermore, we show that glucose (a monomer) is not quite as effective a destabilizer as trehalose (a dimer). The implications of these results related to trehalose-destabilization of DNA, related to conducting and analyzing DNA-additive experiments, and related to using this type of data for predictive purposes are discussed.
Hart
Jonathan L
JL
Department of Chemistry, University of Kentucky, Lexington, KY, USA.
Harris
Zachary M
ZM
Testa
Stephen M
SM
D001482:Base Composition; D002021:Buffers; D004247:DNA / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005947:Glucose / Q000737:chemistry / Q000494:pharmacology; D015394:Molecular Structure; D009690:Nucleic Acid Conformation / Q000187:drug effects; D009696:Nucleic Acids / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D044366:Transition Temperature / Q000187:drug effects; D014199:Trehalose / Q000737:chemistry* / Q000494:pharmacology
D002021:Buffers; D009696:Nucleic Acids; D004247:DNA; D014199:Trehalose; D005947:Glucose