Intrastrand Photo-Crosslinking\nof 5‑Fluoro-2′-O-methyl-4-thiouridine-Modified\nOligonucleotides and Its Implication for Fluorescence-Based Detection\nof DNA Sequences

DNA photo-crosslinking reactions occur widely in biological\nsystems\nand are often used as valuable tools in molecular biology. In this\narticle, we demonstrate the application of an oligonucleotide 5-fluoro-2′-O-methyl-4-thiouridine\n(<b><sup>FS</sup>U</b>)-containing probe for the fluorescent\ndetection of specific DNA sequences. The design of the probe was predicated\non studies of agents that could adversely affect its efficiency. The\nmost important of these is the intrastrand photo-crosslinking of single-stranded\noligodeoxynucleotides bearing <sup><b>FS</b></sup><b>U</b>. Our research findings indicate that <sup><b>FS</b></sup><b>U</b> after photoexcitation can react with nonadjacent bases;\nspecifically, it can react with distant thymine and cytosine residues\nin the chain, forming fluorescent and nonfluorescent intrastrand crosslinks,\nrespectively. In addition, partial photooxidation of the <sup><b>FS</b></sup><b>U</b> residue to 5-fluorouridine was also\nobserved. The results of the study are significant in terms of the\nuse of <sup><b>FS</b></sup><b>U</b>-labeled oligonucleotide\nprobes in the fluorescence-based detection of specific DNA sequences\nbecause the creation of a fluorescent intrastrand crosslink could\nproduce a false signal. To overcome this problem, replacing thymidine\nwith deoxyuridine in the <sup><b>FS</b></sup><b>U</b>-labeled\noligonucleotide probe is proposed and tested.