Advancing small ligands targeting RNA for better binding affinity and specificity: A study of structural influence through molecular design approach

Abstract The cell permeable small fluorescent organic molecules possessing RNA-specific binding property were synthesized for the study of structural influence on two determining factors of binding affinity and specificity. Similar investigations are scarce in the field of RNA-specific binding ligand development based on a systematic structural change of small molecules. The newly developed and structurally simple ligands show much higher RNA-binding affinity (equilibrium binding constant: PI1 , K = 4.50 × 10 5  M −1 ; PI2 , K = 4.48 × 10 5  M −1 ) than that of a well-studied RNA-selective dye E36 (K = 0.89 × 10 5  M −1 ) by the introduction of an aminoethylpiperidine group on the quinolinium scaffold. In addition, the in vivo study of live cell imaging (human prostate cancer cells) indicated that these small molecules are able to provide strong interaction signals when binding with RNA in the region of nucleolus and cytoplasm. The interaction specificity of the molecule towards RNA is found much higher than other types of nucleic acids, which was further supported by the digest tests with ribonuclease (RNase). The experimental results of the present study not only provide important cues on the fluorescent signaling, binding affinity, and specificity advancement with the small RNA-specific binding ligands through structurally refinement of molecular scaffolds, but also give complementary information in searching lead ligands for targeting RNA sequence using high-throughput compound screening techniques and molecular modeling studies.