A ratiometric fluorescence RRE RNA-targeted assay for a new fluorescence ligand

The Rev protein regulates HIV-1 gene expression. Small ligands that bind to the Rev response element (RRE) RNA would inhibit Rev function and suppress HIV-1 replication. A novel ratiometric fluorescence assay was applied in the present study to monitor ligand-RNA interactions by using red-emitting CdTe quantum dots (QDs) coated with silica as a reference. A small fluorescence ligand, ICR 191, was found to interact with RRE at the Rev binding site and compete with the Rev peptide. After adding red-emitting QDs to the interaction system, it was observed that ICR 191 did not fluoresce upon the addition of RRE, and fluorescence recovered when ICR 191 was displaced by a Rev model peptide, whereas the fluorescence of QDs remained constant. Furthermore, variations in the fluorescence ratios between ICR 191 and QDs were exploited to characterize the interactions of Rev with two known antagonists, neomycin B and tobramycin, by using RRE RNA with ICR 191 as a fluorescence indicator. Together, our results demonstrated that ratiometric fluorescence-based nanotechnology applications can be used for ligand-RNA interaction assays. This ICR 191-RRE RNA interaction assay can potentially be developed to build a screening model for assessing antagonists of the Rev binding element in RRE.