Quantitative Categorization of Functional Dynamics of RNA Aptamers by Volume Change

Nucleic acids act as functional materials by coupling the binding of small molecules with dynamic conformational changes. Although the characterization of the dynamics is informative for designing the aptamer and riboswitch, quantitative knowledge of the relationship between the function of nucleic acids and conformational dynamics has been limited. Analysis of the activity of a biomolecule as a function of pressure yields volumetric parameters, which provide information on conformational dynamics. Here, we investigated the binding of flavin mononucleotide (FMN) to the aptamer domain of the FMN riboswitch from Fusobacterium nucleatum under high pressure to quantitatively analyze the volume change (ΔV) during the complex formation. We show that the binding constant (K _a) at 25 °C between FMN and the FMN aptamer decreased with increasing pressure (1.21 × 10⁶ M^-1 at 0.1 MPa to 1.36 × 10⁵ M^-1 at 60 MPa). The ΔV of formation for the FMN/aptamer complex was 87.2 cm³ mol^-1, which was much larger than the value for DNA duplex formation. Analyses of FMN aptamers with mutations in the stem regions showed that a high concentration of Mg²⁺ ions and poly-(ethylene glycol), the latter as a crowding cosolute, decreased the ΔV drastically and synergistically. Our results demonstrate that both an excluded volume effect, occurring due to the exclusion of water during fluctuations of the aptamer before FMN binding, and dehydration upon FMN binding determine the function of the FMN riboswitch acting as a conformational selection model. Thus, analyses of volumetric parameters can be used to quantify the conformational dynamics of nucleic acids depending on their functions.