LAUSR

Nucleoside intercalator conjugates (NICs) describe an innovative methodology developed in our research group for preparation of fluorescence turn‐on DNA hybridization probes targeting specific mRNA sequences (e.g., breast cancer markers). In this methodology, we conjugate a non‐fluorescent intercalator to the base of a nucleic acid (e.g., uracil) via a flexible spacer. This modified monomer can be incorporated into oligonucleotides by solid‐phase synthesis and a large fluorescence enhancement is observed when the modified oligonucleotide is hybridized with its complementary strand due to intercalation of the fluorophore between the two strands. 5‐(6‐p‐Methoxybenzylidene imidazolinone‐1‐hexene)‐2′‐deoxyuridine (dUMBI) is a synthetic monomer to which 4‐methoxybenzylidene imidazolinone (MBI), the fluorescent chromophore of green fluorescent protein (GFP), has been conjugated via a flexible spacer. The detection of human epidermal growth factor receptor 2 (HER2) mRNA by this probe has already been established by our group. The fluorescent intensity of the single‐strand DNA can be considered as negligible due to the free rotation of the fluorophore. Upon hybridization, however, the flexible spacer allows for the intercalation of the fluorophore between the hybridized strands, giving rise to enhanced fluorescence and indicating the presence of target mRNA. 3,5‐Difluoro‐4‐methoxybenzylidene (DFMBI) has enhanced photophysical properties compared to MBI fluorophore. This protocol describes a simple, reliable, efficient, and general method for the synthesis of improved derivative dUDFMBI as a monomer of fluorescent turn‐on DNA hybridization probe with application for detection of HER2 mRNA. © 2020 by John Wiley & Sons, Inc.