Protonation of trimethylamine N-oxide (TMAO) is required for stabilization of RNA tertiary structure

- Trimethylamine N-oxide (TMAO) is known to stabilize RNA tertiary structure.
- Constant pH MD simulations indicate that RNA leads to the presence of protonated TMAO.
- The presence of protonated TMAO is shown to stabilize the tertiary structure of RNA based on simulations of the PreQ1 riboswitch.
- Protonated TMAO stabilizes the RNA by dehydrating the phosphodiester backbone and mimicking the behavior of ions.

The osmolyte trimethylamine N-oxide (TMAO) stabilizes the tertiary but not the secondary structures of RNA. However, molecular dynamics simulations performed on the PreQ1 riboswitch showed that TMAO destabilizes the tertiary riboswitch structure, leading us to hypothesize that the presence of RNA could result in enhanced population of the protonated form, TMAOP. Constant pH replica exchange simulations showed that a percentage of TMAO is indeed protonated, thus contributing to the stability of the tertiary but not the secondary structure of PreQ1. TMAOP results in an unfavorable dehydration of phosphodiester backbone, which is compensated by electrostatic attraction between TMAOP and the phosphate groups. In addition, TMAOP interacts with specific sites in the tertiary RNA structure, mimicking the behavior of positively charged ions and of the PreQ1 ligand in stabilizing RNA. Finally, we predict that TMAO-induced stabilization of RNA tertiary structures should be strongly pH dependent.