A General SAXS-Based Screening Protocol Validated in RNA-Protein Interactions

Abstract

RNA-protein interactions can be characterized by both sequence-specific and non-specific interactions, due to the ubiquitous presence of charged contacts associated with nucleic acid backbones. This multiplicity of binding modes complicates screening efforts to identify strong-affinity RNA sequences (also known as cognate motifs,) required to apply structural biology techniques. In addition, the cost of production of specific RNA sequences hinder current efforts using techniques such as ITC and NMR to validate motifs enriched from affinity screens. We report a screening method based on small-angle X-ray scattering (SAXScreen) that summarises a ligand's efficacy at eliciting signal changes as SΔ, a structural analogue to affinity KD that also captures receptor-ligand scattering and conformational changes. Since no structure-specific information is required, the protocol can be generalised towards various biomolecular interactions and processes that are detectable via changes in scattering patterns. Utilising synchtron sources and automated sample-changer setups, we validated the approach by conducting sparse-search strategies covering ∼35 oligoneucleotide sequences around the UGUx motif of Sex-lethal protein, yielding both ligand ranking and differentiation between binding mechanisms that are further confirmed in ITC and NMR titrations.