Efficient Thymidine-Selective DNA Interstrand Photo-activated Crosslinking by the 6-Thioguanine Connected via an Ethylene-Linker to the 2′-Deoxyribose Unit

Abstract
Cross-linking is a widely utilized technique in the study of DNA, RNA, and their interactions with proteins. This method involves the incorporation of chemically or enzymatically active alkylating groups and photo-activated agents into nucleic acids. Thionucleobases, which closely mimic natural bases, form cross-links upon exposure to UVA light. These cross-links occur only at sites in close proximity, facilitating the identification of interactions within nucleic acids or between nucleic acids and proteins in complex assemblies. However, thionucleobases are less efficient at cross-linking with their natural base counterparts in a DNA duplex. In this study, 6-thioguanine was linked to 2′-deoxyribose through an ethylene spacer at the 1′-position (Et-thioG), aiming to position the 6-thio group near the complementary nucleobase on the opposite strand. In duplexes where 2′-deoxy-6-thioguanosine (6-thio-dG) did not form a cross-link, Et-thioG effectively cross-linked with thymine (T) with high selectivity upon UVA exposure, but showed much lower efficiency with adenine (dA), guanine (dG), cytosine (dC), 5-methyl-cytosine (5-methyl-dC), or uracil (dU). Notably, the yield of the photo-crosslinked product with thymine improved significantly in the presence of dithiothreitol or sodium hydrosulfide (NaSH) at low UVA doses. This efficient and selective cross-linking at reduced UVA doses could be advantageous for biological applications of 6-Thio-dG Et-thioG.