Ligand “switching on” fluorescence of HIV-1 RNA-templated copper nanoclusters for ligand–RNA interaction assays

Ligand–RNA interaction assay provides the basis for developing new RNA-binding small molecules. In this study, fluorescent copper nanoclusters (CuNCs) were first prepared using two kinds of HIV-1 RNA targets, rev-responsive element (RRE) and transactivator response element (TAR) RNA, as new templates, and it was found that the fluorescence of the single RNA-templated CuNCs was negligible. Using neomycin as a model drug, the fluorescence could be augmented (approximately 6 times) for the neomycin/RNA-templated CuNCs. Thus, a novel method was developed for ligand–RNA interactions by observing the fluorescence changes in CuNCs prepared using RNA before and after the addition of ligands. The preparation parameters of neomycin/RNA-CuNCs were optimized. The as-prepared CuNCs were characterized using UV–vis spectroscopy, fluorescence spectroscopy, and high-resolution transmission electron microscope. Circular dichroism spectral analysis showed that RRE and TAR were inclined to form a double-stranded structure after interaction with neomycin, which was more conducive to the formation of CuNCs. The interactions of neomycin and three test drugs (amikacin, gentamicin, and tobramycin) with RNA were investigated using the proposed method, and the binding constants and number of binding sites were obtained through theoretical calculations. This study provides a novel approach for ligand–RNA interaction assays.