Inhibition of dicing of guanosine-rich shRNAs by quadruplex-binding compounds

Abstract

RNA interference is triggered by small hairpin precursors that are processed by the endonuclease dicer to yield active species such as siRNAs and miRNAs. To regulate the RNAi-mediated suppression of gene expression, we imagined a strategy that relies on the sequence-specific inhibition of shRNA precursor processing by immediate RNA-small molecule interactions. Here, we present a first step in this direction by augmenting shRNAs with guanosine-rich sequences that are prone to fold into four-stranded structures. The addition of small molecules that selectively bind to such quadruplex sequences should allow for the specific inhibition of dicing of shRNAs that contain suitable G-rich elements. In an attempt to find compounds that protect against dicer processing, we have examined the effects of quadruplex-binding compounds on the dicer processing of shRNAs containing G-quadruplexes. Although a variety of small molecules that are known to bind to quadruplexes inhibited in vitro dicing of shRNAs, only two substance classes, namely certain porphyrazines and bisquinolinium compounds, showed selective inhibition of G-rich shRNAs compared to control sequences lacking guanine-rich elements. The G-rich shRNAs displayed a potent knockdown of gene expression in mammalian cell culture, but the effect was not influenced by addition of the respective quadruplex-binding compounds.