RNA Photolabeling Mechanistic Studies: X-ray Crystal Structure of the Photoproduct Formed between 4-Thiothymidine and Adenosine upon Near UV Irradiation

摘要

In recent years, reliable photolabeling methods have been developed to probe RNA tertiary structure in solution. One of these exploits the remarkable photochemical properties of the sulfur analogs of the current nucleic acid bases 4-thiouracil, 4-thiothymine, and 6-mercaptopurine to gain informative data on tertiary interactions within such biomolecules. Indeed, the latter bases in which an oxygen has been replaced by sulfur are stable in the dark and can be selectively photoactivated to give highlyreactive excited states manifesting the capacity to undergo covalent bonding with any nucleic acid base. Hence, when a thio-substituted nucleobase is introduced either ramdomly or at a defined position in such a system (by application of appropriate enzymatic or chemical methods), subsequent irradiation usually results in the formation of informative crosslinks. Obviously, only residues located at bonding distance with the thio-inodified nucleobase can be involved in the cross-links which can be mappedby suitable sequencing procedures. Interestingly, the spatial relationships which are evidenced in this manner can serve as constraints for the reconstruction of the three-dimensional structure of the system and the analysis of its conformational flexibility by molecular modeling.6 Furthermore, the respective orientation of the two residues leading to a cross-link can be more precisely defined when the corresponding photochemical pathways leading to the products have been elucidated. In general, these pathways can be reasonably deduced from the structural analysis of the photo-products. So far, detailed structural analysis of photoproducts resulting from thio-substituted nucleobases has been accomplished with pyrimidine only.7 As photo-cross-links involving purine residues are frequently encountered in the analysis of RNA folding,5 we describe here the X-ray crystal structure of the unique photoproduct8 which was formed upon irradiation (ca. 360 nm, inert atmosphere) of an equimolecular mixture (8.6 mM) of 4-thiothymidine (1) and adenosine (2) in water.