Gas-phase stabilization by tertiary interactions in yeast tRNA^Phe and quaternary contacts in a small RNA-RNA complex

Native mass spectrometry (nMS) and ion mobility (IM) are now established tools for the structural analysis of proteins and protein complexes, but their application to RNAs and RNA-RNA assemblies remains less developed. Here, we examine how magnesium, ionization polarity, solution temperature, and collisional activation influence the mass-corrected collision cross section (mCCS) of wild-type (WT) and a structurally destabilized mutant (MT) yeast tRNA^Phe. Under positive-polarity ionization, magnesium was essential for WT tRNA^Phe to fold into and retain native-like structures. In 2 mM magnesium acetate, increasing the solution temperature from 25 to 60°C produced little change in the mCCS of WT tRNA^Phe, whereas the mCCS of MT tRNA^Phe increased. Collisional activation further distinguished the two constructs: increasing the trap collision energy induced a pronounced decrease in the mCCS of WT but only a modest decrease for MT tRNA^Phe, consistent with the absence of magnesium-supported tertiary contacts in the mutant. We also assessed the HJ3 RNA-RNA heterodimer using collision- and surface-induced dissociation (CID and SID, respectively). CID primarily resulted in charge stripping, while SID generated proportionally charged monomers, indicating preservation of native quaternary contacts. Collectively, these results demonstrate how IM-nMS measurements can be leveraged to gain insights into the tertiary and quaternary RNA structures and their gas-phase stability.