The nucleosidic photolabels 2-5, able to form long-range photo-cross-links, have been used to further investigate the tertiary folding of a hammerhead ribozyme domain. These photolabels derive from 2 H -deoxyuridine which is substituted at its C-5 position with a photoactivable 4-thiothymine unit linked by a chain of various length and rigidity. Derivatives 2-5 were inserted at strategic positions of a deoxysubstrate analogue (dS) of a hammerhead ribozyme (Rz). Covalent cross-links were generated by 366 nm irradiation of the Rz-dS complexes assembled under cleavage conditions. The Rz residues involved in these cross-links were mapped to give new sets of proximity data extending those previously obtained by intrinsic photolabelling with 2 H -deoxy-4-thiouridine 1. Thus, compared to the zero-length crosslinker 1, photolabels 2-5 exhibit a higher exploration capacity which depends critically upon the length and flexibility of the linker and their site of incorporation. Interestingly, in contrast to 1, these photolabels were able to cross-link residues involved in the double G:A mismatch as well as residues of the adjacent base pairs. These findings suggest that the mismatch domain exhibits an unexpected conformational flexibility in solution.