Quantifying the Temperature Dependence of Glycine—Betaine RNA Duplex Destabilization

Abstract

Glycine betaine stabilizes folded protein structure due to its unfavorable thermodynamic interactions with amide oxygen and aliphatic carbon surface area exposed during protein unfolding. However, glycine betaine can attenuate nucleic acid secondary structure stability, although its mechanism of destabilization is not currently understood. In this work we quantify glycine betaine interactions with the surface area exposed during thermal denaturation of nine RNA dodecamer duplexes with guanine-cytosine (GC) contents of 17–100%. Hyperchromicity values indicate increasing glycine betaine molality attenuates stacking. Glycine betaine destabilizes higher GC content RNA duplexes to a greater extent than low GC content duplexes due to greater accumulation at the surface area exposed during unfolding. The accumulation is very sensitive to temperature and displays characteristic entropy-enthalpy compensation. Since the entropic contribution to the m-value (used to quantify GB interaction with the RNA solvent accessible surface area exposed during denaturation) is more dependent on temperature than the enthalpic contribution, higher GC content duplexes with their larger transition temperatures are destabilized to a greater extent than low GC content duplexes. The concentration of glycine betaine at the RNA surface area exposed during unfolding relative to bulk was quantified using the solute partitioning model. Temperature correction predicts a glycine betaine concentration at 25 °C to be nearly independent of GC content, indicating that glycine betaine destabilizes all sequences equally at this temperature.

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

“…It has been shown previously that cosolutes generally destabilize nucleic acid secondary structure via preferential interactions of the cosolute with the nucleobases in the unfolded state ( Lambert & Draper, 2007 ; Knowles et al, 2011 ), and differences in observed m-values are then due to differences in the chemistry of specific interactions of the cosolutes with given sequences. For example, betaine prefers G-C pairs, urea prefers A-T pairs, whereas polyethylene glycols have previously shown no distinct sequence preference ( Nordstrom et al, 2006 ; Spink, Garbett & Chaires, 2007 ; Knowles et al, 2011 ; Schwinefus et al, 2013 ). In determining these dependences, large global changes are often made; whereas for our RNA hairpin model system our sequence changes are small.…”

l-Proline and RNA Duplex m-Value Temperature Dependence

“…Proline was a very strong destabilizer of RNA duplexes, in agreement with previous studies. ,,, The transition temperature at an RNA single strand unfolded fraction of 0.5 in 0 m proline decreased by approximately 5.5 °C to the same unfolded fraction in 1 m proline. Solutes, such as urea and glycine betaine, exhibited transition temperature attenuations of 2.6 °C and 2.7 °C, respectively, with a 12 base pair RNA duplex with similar GC content. Proline’s strong destabilization capability made proline a candidate as a chemical denaturant to fully unfold RNA duplexes, subject to proline’s solubility limit (next section).…”

“…High GC content duplexes were also destabilized to a greater extent in glycine betaine solutions when destabilization was monitored by thermal denaturation. ,, The greater destabilization of GC-rich relative to AT- or AU-rich nucleic acids was attributed to glycine betaine’s “isostabilizing” ability . Glycine betaine’s isostabilizing ability originated from the temperature dependence of glycine betaine interactions with nucleic acid surfaces . Since proline m -values obtained from thermal denaturation were obtained at temperatures well above room temperature, any temperature dependence of proline interactions with the unfolded RNA surface area would make comparison of m -values from thermal denaturation and predicted m -values in Figure tenuous at best.…”

“…Proline exhibits similar behavior as glycine betaine, preferentially destabilizing GC-rich nucleic acid secondary structure to a greater extent than AT- or AU-rich duplexes . Recently, the m -value for glycine betaine interactions with nucleic acid surface area was shown to be temperature dependent . For proline to serve as a probe of biopolymer conformational changes, the temperature dependence of proline interactions with biopolymer solvent accessible surface area must be elucidated since biochemical reactions in vitro are often executed at temperatures other than room temperature.…”

Advanced Evanescent-Wave Optical Biosensors for the Detection of Nucleic Acids: An Analytic Perspective

“…Tetramethylamonium chloride (TMAC) and glycine betaine have been reported to be isostabilizing agents, altering the melting temperature and making the hybridization solely dependent on oligonucleotide length, despite their GC content (Duby et al, 2004; Napolitano et al, 2004; Schwinefus et al, 2007). The destabilizing effect of such agents increases with increasing GC content, with almost no effect on poly(dAdT) (Schwinefus et al, 2013). On the other hand, some other components, such as urea, have the opposite effect, promoting a decrease in the strength of AT/U bonds more than GC base pairs (Schwinefus et al, 2007).…”