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.

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CALI and the labelling specificity that fluorescent proteins provide is very useful to avoid uncontrolled photodamage. Indeed, fluorescent proteins have been successfully used in CALI, although of the inactivation mechanisms by ROS are dependent on the fluorescent protein used and are not fully understood [2,3]. Here, we present a quantitative study of the ability of TagRFP to produce ROS, in particular singlet oxygen. TagRFP is able to photosensitize singlet oxygen with an estimated quantum yield of 0.004 [4]. This is the first estimation of a quantum yield of singlet oxygen production value for a GFP-like protein. We also find that TagRFP has a short triplet lifetime, which reflects relatively high oxygen accessibility to the chromophore compared to EGFP. Our results provide photophysical insight that allows the understanding of the mechanism behind CALI. Moreover, it has implications in improving photobleaching in fluorescent proteins. [1] K.