A "pseudo" fluorescence indicator displacement assay based on "light-up" sanguinarine for identifying small ligands targeting HIV-1 RRE RNA

Abstract

Developing effective methods to identify drugs that can target HIV-1 Rev. response element (RRE) RNA and block the interaction between Rev. and RRE has practical significance in the treatment of AIDS. Fluorescence indicator displacement (FID) assay was commonly employed to identify small ligands binding to RNA. In this study, the non-fluorescent sanguinarine (Sang) was used as a novel "pseudo" fluorescence indicator to identify small ligands targeting RRE through its fluorescence "light-up", assisted by β-cyclodextrin (β-CD) or nano-SiO₂. The fluorescence enhancement effect of β-CD or nano-SiO₂ on Sang was initially examined. Subsequently, the Sang-RRE interaction was investigated and validated using circular dichroism, and the binding constants and binding sites of Sang to RRE were obtained; the competition between Sang and Rev. was explored and confirmed through molecular docking. Finally, the experimental parameters of the "pseudo" FID assay including reaction time, along with β-CD and nano-SiO₂ concentrations, were optimized using a well-known Rev. inhibitor (neomycin). Under the optimized experimental conditions, the evaluation of three positive inhibitors and one negative control was performed. These findings presented a novel approach for identifying small ligands targeting HIV-1 RRE RNA using a non-fluorescent ligand in the FID assay and established a promising screening platform.

Keywords: Fluorescence indicator displacement assay; RRE RNA; Sanguinarine.