2016 Volume 64 Issue 9 Pages 1315-1320
Cross-linking is a widely-used technology in the studies of DNA, RNA and their complexes with proteins. Intrinsically active alkylating moieties and photo-activated agents are chemically or enzymatically incorporated into nucleic acids. Thionucleobases resemble the corresponding natural bases, and form cross-links by UVA irradiation. They form cross-links only with a site in close contact, thereby allowing identification of the contacts within the nucleic acids and/or between the nucleic acids and proteins in complex nucleoprotein assemblies. On the other hand, the thionucleobase forms a cross-link less efficiently for the reaction with the opposite natural base in the DNA duplex. In this study, 6-thioguanine was connected to 2′-deoxyribose through an ethylene linker at the 1′-position (Et-thioG). The linker was expected to bring the 6-thio group close to the nucleobase in the opposite strand. In a duplex in which the 2′-deoxy-6-thioguanosine (6-thio-dG) did not form a crosslink, Et-thioG efficiently formed crosslink with a high selectivity for T by UVA irradiation, but with a much lower efficiency for dA, dG, dC, 5-methyl-dC or dU. Interestingly, the yield of the photo-crosslinked product with dT was effectively improved in the presence of dithiothreitol or sodium hydrosulfide (NaSH) at a low UVA irradiation dose. The efficient and selective cross-link formation at a low UVA dose may be beneficial for the biological application of Et-thioG.
