Synthesis of 4-Thiothymine Based Photolabels as New Tools for Nucleic Acids Structural Studies in Solution: Formation of Long-Range Photo-Cross-Links within a Hammerhead Ribozyme Domain

Abstract

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 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′-deoxy-4-thiouridine 1. Thus, compared to the zero-length cross-linker 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.