Methods for using new conceptual tools and parameters to assess RNA structure by small-angle X-ray scattering

Understanding the biological activities of riboswitches and of RNA in general requires a thorough analysis of both the spatial arrangement of the residues and the dynamics of the structural ensemble. Specifically, evaluating the structural basis for riboswitch function requires analyses of many relevant states that include ligand-bound and -free, high Mg(2+), and quite possibly, the active transcription state, which is challenging to achieve by most methods. Small angle X-ray scattering (SAXS) is an enabling technique for comprehensive analyses of RNA structures in solution. Here, we describe recent SAXS tools and technologies that substantially improve the potential for accurate and comprehensive analyses of flexibility, unstructured elements, conformational selection, and induced fit in RNA function. We note equipment needed plus appropriate annealing and purification procedures. We describe key model-independent parameters (SAXS invariants) which can be used to monitor changes in a particle's thermodynamic state: the Guinier-based Rg, the volume-of-correlation (Vc), the Porod-Debye exponent (P(E)), and the power-law parameter, Q(R), that determines mass directly from the SAXS data. We also consider the value of real-space parameters and of multiphase modeling with MONSA to locate secondary structure elements within SAXS volumetric envelopes. For conformation changes, experiments with nanogold-labeled RNA analyzed using the SAXS structural comparison map and volatility ratio difference metric enable high-throughput evaluation of solution-state conformations. Collectively, the described tools and procedures enable quantitative and comprehensive measures of riboswitch structures with general implications for our views and strategies of RNA structural analysis.