A small-molecule fluorescence probe ANP77 for sensing RNA internal loop of C, U and A/CC motifs and their binding molecules

Small-molecules interacting with particular RNAs and modulating their functions are vital tools for RNA-targeting drug discovery. Considering the substantial distribution of the internal loops involving two contiguous cytosines opposite to a single-nucleotide base (Y/CC; Y = C, U or A) within the biologically significant functional RNAs, developing small-molecule probes targeting Y/CC sites should provide profound insight into their functions and roles in biochemical processes. Herein, we report ANP77 as the small-molecule probe for sensing RNA internal loop of Y/CC motifs and molecules binding to the motifs. The Y/CC motifs interact with ANP77 via the formation of a 1:1 complex and quench the fluorescence of ANP77. The flanking sequence-dependent binding to C/CC and U/CC sites was assessed by fluorometric screening, provided the binding heat maps. The quenching phenomena of ANP77 fluorescence was confirmed with intrinsic potential drug target pre-miR-1908. Finally, the binding-dependent fluorescence quenching of ANP77 was utilized in the fluorescence indicator displacement assay to demonstrate the potential of ANP77 as an indicator by using the RNA-binding drugs, risdiplam and branaplam.