Fluorescent protein probe detects parallel hetero G-quadruplex induced by G-rich ASO for gene silencing

Hetero G-quadruplex assembly by RNA and G-rich antisense oligonucleotide (G-ASO) has recently emerged as a potential approach for regulating gene expression. In investigating the assembly of a hetero G-quadruplex-inducing G-ASO1, we propose a simple fluorescent protein probe for the detection and determination of hetero G-quadruplex formation. The fluorescent protein probe was genetically generated by fusing the RHAU domain to cyan fluorescent protein (CFP). Moreover, the hetero G-quadruplex-inducing G-ASO1 was also applied to regulate the expression of a model enhanced green fluorescent protein (G-eGFP) in vitro. Fluorescence analysis using the RHAU-CFP probe showed that the hetero G-quadruplex-inducing G-ASO1 generated a fluorescence signal approximately 35-fold and 12-fold higher than those of Htelo2 (a non-parallel G-quadruplex) and G-ASO1 alone, respectively. These results demonstrate that the hetero G-quadruplex-inducing G-ASO1 was arranged into a parallel hetero G-quadruplex topology. In addition, a 400 nM G-ASO1 concentration was able to significantly inhibit around 75% of the expression of the model protein G-eGFP in vitro, whereas ASO2, which lacks G-rich sequences, inhibited around 30% of protein expression under the same conditions. This fluorescent protein probe provides a simple and effective tool for the screening and determination of hetero G-quadruplex-inducing G-ASO in biomedical applications.