A general small-angle X-ray scattering-based screening protocol validated for protein-RNA interactions

Chen P1, Masiewicz P1, Rybin V1, Svergun D2 and Hennig J1

  1. EMBL Heidelberg, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
  2. 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.