A general small-angle X-ray scattering-based screening protocol validated for protein-RNA interactions
- EMBL Heidelberg, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
- EMBL Hamburg, DESY, Building 25A, Notkestrasse 85, 22607 Hamburg, Germany.
We present a screening protocol utilizing small-angle X-ray scattering (SAXS) to obtain structural informatio on biomolecular interactions independent of prior knowledge, so as to complement affinity-based screening and provide leads for further exploration. This protocol categorizes ligand titrations by computing pairwise agreement between curves, and separately estimates affinities by quantifying complex formation as a departure from the linear sum properties of solution SAXS. The protocol is validated by sparse sequence search around the native poly uridine RNA motifs of the two-RRM domain Sex-lethal protein (Sxl). The screening of 35 RNA motifs between 4 to 10 nucleotides reveals a strong variation of resulting complexes, revealed to be preference-switching between 1:1 and 2:2 binding stoichiometries upon addition of structural modeling. Validation of select sequences in isothermal calorimetry and NMR titration retrieves domain-specific roles and function of a guanine anchor. These findings reinforce the suitability of SAXS as a complement in lead identification. In addition to the published data, we intend to present current progress in determining performance limits for test systems at ESRF Grenoble and DESY Hamburg.