Synthesis of 4-thiothymine based photolabels as new tools for nucleic acids structural studies in solution: Formation of long-range photo-cross-linkswithin a hammerhead ribozyme domain

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'-deoxyuridine which is su bstituted at its C-5 position with a photoactivable 4-thiothymine unit link ed by a chain of various length and rigidity. Derivatives 2-5 were inserted at strategic positions of a deoxysubstrate analogue (dS) of a hammerhead r ibozyme (Rz). Covalent cross-links were generated by 366 nm irradiation of the Rz-dS complexes assembled under cleavage conditions. The Rz residues in volved in these cross-links were mapped to give new sets of proximity data extending those previously obtained by intrinsic photolabelling with 2'-deo xy-4-thiouridine 1. Thus, compared to the zero-length cross-linker , photol abels 2-5 exhibit a higher exploration capacity which depends critically up on the length and flexibility of the Linker and their site of incorporation . Interestingly, in contrast to 1, these photolabels were able to cross-lin k residues involved in the double G:A mismatch as well as residues of the a djacent base pairs. These findings suggest that the mismatch domain exhibit s an unexpected conformational flexibility in solution. (C) 2000 Elsevier S cience Ltd. All rights reserved.