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.

A comparison of DNA and RNA quadruplex structures and stabilities

“…18,19 In a further study, we used quadruplexes as small-molecule-addressable sequences for the regulation of dicer-mediated processing of G-rich shRNAs. 32 In order to better judge the stabilities of RNA quadruplexes, we have carried out a comparative study by investigating DNA and RNA quadruplex sequences in the absence and presence of the monovalent ions potassium and sodium which are known to stabilize quadruplexes by coordination with the 6-oxo-functionality of the tetrad-forming guanosines.…”

Chemical Biology Approach for Dissection of RNAi/miRNA Pathway

“…While technically challenged, the success in identification of miR-21-specific inhibitor could shed light on the feasibility of the chemical biology approach in this regard [39]. Besides potentially targeting the protein(s) involved in the regulation of selective miRNAs processing, several small molecules could directly bind to RNAs, including miRNAs [42][43][44][45]. Given the diversity of RNA secondary structures among miRNA precursor population, it is plausible to speculate that small molecules targeting specific pri-, pre-or mature miRNA could be identified.…”

Small Molecule Modifiers of the microRNA and RNA Interference Pathway

“…Several small molecules have been reported that directly bind to RNAs (65,66), including miRNAs (67,68). Helixthreading peptides were discovered, which consist of an acridine core that acts as a RNA-duplex intercalator, which is flanked by short peptides (e.g., N-Ser-Val-acridine-Arg-C) (67).…”

“…However, no pre-miR-39 binding of the compound has been demonstrated to date. In another report, an shRNA-targeting luciferase was modified with a G-rich loop and three additional Gs at the 5′ end, in order to enable potential G-quadruplex formation (68). Even though no evidence for actual G-quadruplex formation of this oligomer was provided, a set of classical G-quadruplex small molecule binders were screened for their ability to inhibit processing of the shRNA.…”