Fluorogenic indicator for targeting HIV-1 TAR RNA: Evaluation for binging and signaling functions of monomethine cyanine dye thiazole orange and its application to FID assay

Thiazole orange (TO), a typical monomethine cyanine dye, does work as a useful fluorogenic indicator in FID (fluorescent indicator displacement) assay for targeting HIV-1 TAR (transactivation responsive region) RNA. A comparison of the binding and signaling functions of a series of monomethine cyanine dyes, including TO-PRO1, YO-PRO-1 JO-PRO-1 and BO, reveals that TO is the best indicator for TAR RNA among these dyes. The binding of TO is highly selective to TAR RNA over duplex RNA, and the dissociation constant, K_d, reaches 60±7 nM (pH 6.5, 10 mM sodium phosphate, 50 mM NaCl, 0.1 mM EDTA, 5°C). The binding to TAR RNA is accompanied by a significant light-up response (f_free < 0.01, f_bound = 0.198), where the light-up factor, I/I₀ (I and I₀ denote the fluorescence intensities in the presence and absence of the target RNA, respectively), reaches ca. 370-fold (λ_ex = 501.5 nm, λ_em = 530.4 nm). In addition, an examination of the Z'-factor in a 384-well microplate format resulted in a value of > 0.5, revealing the high quality of the TO-based assay for HTS (high throughput screening). These promising functions of TO are discussed as a rational basis for the advanced design of RNA-binding indicators for FID assay that targets HIV-1 TAR RNA.