Intrastrand Photocrosslinking of 5-Fluoro-2’-O-Methyl-4-Thiouridine Modified Oligonucleotides and its Implication for Fluorescence-Based Detection of DNA Sequences

Abstract

Abstract DNA photocrosslinking reactions occur widely in biological systems and are often used as a valuable tool in molecular biology. Herein we report the sequence and temperature-dependent intrastrand photocrosslinking of single-stranded oligodeoxynucleotides bearing 5-fluoro-4-thiouridine (FSU). Our research findings indicate that FSU can photoreact with non-adjacent bases, specifically, it can react with distant thymine and cytosine residues in the chain forming fluorescent and non-fluorescent intrastrand crosslinks, respectively. In addition, partial photooxidation of FSU residue to 5-fluorouridine was also observed. The results of the study are significant in terms of the use of FSU-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 FSU-labeled oligonucleotide probe is proposed and tested.