Here, we have investigated in systematic fashion the structural requirements for the cross-linking of an RNA to its RBP partner. We used the RRM domain of the RBFOX family (FOXRRM) and its RNA consensus binding motif U1G2C3A4U5G6U7 (FOXRBE) as a model system because the complex forms with high affinity, the RRM is a highly abundant RNA binding domain and its structure has been well characterized . We introduced C-labelled ribonucleotides systematically into the FOXRBE heptanucleotide using solid-phase synthesis and used CLIR-MS to identify RNA-protein cross-links with site-specific resolution. High-yielding cross-linking was localised at two clusters of amino acids around two phenylalanines, consistent with previous findings (Götze et al, submitted). However, with few exceptions, it only occured at U1, G2 and G6. We then employed site-specific mutagenesis of both the RNA and the protein to probe the influence of nucleotide species, its position in the RNA sequence and amino acid composition on cross-linking. For each RNA-protein pair, we first confirmed that the same binding mode was maintained as in the wild-type interaction. The analysis revealed that: i) highyielding cross-linking only occurred at three of the seven nucleotide positions and involved guanosine or uridine, and that guanine and uracil were not interchangeable; ii) only nucleobases that stacked with the aromatic amino acid side chains reacted strongly; and, surprisingly, iii) the primary stacking interaction was a requirement for neighbouring amino acids to react. We confirmed the importance of this primary stacking feature in other published studies for which cross-linking and structural data are available. The analysis strongly suggested that a stacking interaction is required to activate a nucleotide for free-radical-based cross-linking reactions in native RNA-protein complexes. We expect that this finding will facilitate the interpretation of RNA-protein cross-linking data. Moreover, it will aid the development of new tools for de novo motif discovery (see refs in ) especially for non-canonical binding motifs, and it will help guide the design of future cross-linking experiments and methods.