Analyzing and predicting the thermodynamic effects of the metabolite trehalose on nucleic acids

Abstract

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.

Comprehensive evaluation of molecular enhancers of the isothermal exponential amplification reaction

“…Briefly, under current EXPAR conditions, 1.8 M ethylene glycol, 0.8 M propylene glycol, 1 M betaine, 5% DMSO, 0.4 M trehalose and 40 mM TMAC lowered the Tm between target and template by at least 2 °C. Each of those small molecules decreased Tm to different degrees, consistent with the literature152232333435. In contrast, 1 mg/mL BSA and 10 μg/mL SSB proteins had minimal effect on Tm (1 °C or less).…”

Trehalose facilitates DNA melting: a single-molecule optical tweezers study

“…9 Under similar buffer conditions as ours in bulk assays, the zero-force melting temperature of dsDNA molecules with similar GC content was found to decrease by 4.4 C per molar concentration of trehalose. 10 The extent of this change was found to depend strongly on the GC content but only minimally on the DNA length. 10 Over the range of trehalose concentrations assayed here (from 0 to 100 mM), these results would imply a decrease of the order of 1 C in the melting temperature (T 0 m ) and in T Fos m in our experiments.…”

“…S3 †), that is consistent with more pronounced effect of trehalose on GC DNA. 10 We also analyzed the effect of trehalose on the surrounding aqueous environment by calculating the radial distribution function (RDF) for water molecules and DNA in the presence and in the absence of trehalose in the simulation box (Fig. S4 †).…”

“…9 The extent of DNA destabilization due to trehalose depends on DNA length and on percent guanine-cytosine (GC) content as concluded from systematic thermal experiments on short duplexes. 10 While the evidence for the inuence of trehalose on proteins and nucleic acids is compelling, our understanding of the underlying mechanism is incomplete. Various mechanisms have been considered to explain the effect of trehalose on biomolecules, 1 including physically shielding parts of biomolecules and removing the hydration waters by replacing (direct interaction with biomolecule) or sequestering them (no direct interaction with biomolecule).…”

Effects of osmolytes and macromolecular crowders on stable GAAA tetraloops and their preference for a CG closing base pair

“…In contrast to their effects on protein structures, the effects of macromolecular crowders and osmolytes on nucleic acid structures have been shown to be rather complex and have significant dependencies on the chemical and structural properties of both the nucleic acid and the cosolute ( Spink & Chaires, 1999 ; Lambert & Draper, 2007 ; Downey et al, 2007 ; Nakano et al, 2012 ; Nakano et al, 2008 ; Nakano et al, 2009 ; Kilburn et al, 2010 ; Lambert, Leipply & Draper, 2010 ; Hart, Harris & Testa, 2010 ; Knowles et al, 2011 ; Blose et al, 2011 ; Pramanik et al, 2011 ; Gu et al, 2013 ). Osmolytes and crowders have been shown repeatedly to destabilize some secondary structures ( Spink & Chaires, 1999 ; Lambert & Draper, 2007 ; Nakano et al, 2008 ; Nakano et al, 2012 ; Lambert, Leipply & Draper, 2010 ; Hart, Harris & Testa, 2010 ; Knowles et al, 2011 ; Blose et al, 2011 ; Pramanik et al, 2011 ; Gu et al, 2013 ; Strulson et al, 2013 ) while either stabilizing or destabilizing multi-stranded or more complex, higher-order structures ( Spink & Chaires, 1999 ; Lambert & Draper, 2007 ; Downey et al, 2007 ; Lambert, Leipply & Draper, 2010 ; Strulson et al, 2014 ). Some osmolytes and macromolecular crowders have also been shown to facilitate folding and function of catalytic RNA ( Nakano et al, 2009 ; Nakano et al, 2014 ; Kilburn et al, 2010 ; Strulson et al, 2013 ).…”