Intrastrand Photo-Crosslinking of 5-Fluoro-2′-O-methyl-4-thiouridine-Modified Oligonucleotides and Its Implication for Fluorescence-Based Detection of DNA Sequences

DNA photo-crosslinking reactions occur widely in biological systems and are often used as valuable tools in molecular biology. In this article, we demonstrate the application of an oligonucleotide 5-fluoro-2'-O-methyl-4-thiouridine (<b>^FSU</b>)-containing probe for the fluorescent detection of specific DNA sequences. The design of the probe was predicated on studies of agents that could adversely affect its efficiency. The most important of these is the intrastrand photo-crosslinking of single-stranded oligodeoxynucleotides bearing ^<b>FS</b><b>U</b>. Our research findings indicate that ^<b>FS</b><b>U</b> after photoexcitation can react with nonadjacent bases; specifically, it can react with distant thymine and cytosine residues in the chain, forming fluorescent and nonfluorescent intrastrand crosslinks, respectively. In addition, partial photooxidation of the ^<b>FS</b><b>U</b> residue to 5-fluorouridine was also observed. The results of the study are significant in terms of the use of ^<b>FS</b><b>U</b>-labeled oligonucleotide probes in the fluorescence-based detection of specific DNA sequences because the creation of a fluorescent intrastrand crosslink could produce a false signal. To overcome this problem, replacing thymidine with deoxyuridine in the ^<b>FS</b><b>U</b>-labeled oligonucleotide probe is proposed and tested.