MicroRNAs (miRNAs) regulate gene expression in cells; high levels of expression are associated with various cancers. In this paper, we describe PyA-modified nucleic acid probes that can detect intracellular miRNAs by forming DNA/RNA hybrid three-way junction structures containing a fluorescent scaffold-a so-called G-cluster. This G-cluster featured two mismatched strands, four guanine residues, and one fluorescent adenine residue having a pyrene moiety covalently connected at the 8-position through an acetylene linker. The scaffold underwent a dramatic shift in its emission wavelength when two mismatched strands formed a duplex, similar to the behavior of an adenine pentad system (A-cluster). We applied the G-cluster scaffold in a three-way junction system to probe for miRNAs; its red-shifted fluorescence intensity and stability were greater than those reported previously for A-cluster three-way junction probes. Furthermore, confocal microscopy of cancer cell lines revealed bright fluorescence emissions in response to the miRNAs in the cells. Thus, this system can be applied intracellularly as a potential fluorescent probe for the detection of various biologically important nucleic acids.
Keywords: confocal microscopy; fluorescent probe; guanine; miRNA detection; three-way junction.