RNA-binding landscape of amiloride: large-scale profiling and structural basis of U-U mismatch recognition

Amiloride possesses a characteristic chemical scaffold capable of recognizing uracil (U) through three complementary hydrogen bonds; however, its binding selectivity toward naturally occurring RNA structural motifs has remained uncharacterized. In this study, we present a large-scale analysis of amiloride's RNA binding properties and structural characterization of the amiloride-RNA complex. Using folded RNA element profiling with structure library (FOREST), we evaluated the RNA-binding selectivity of amiloride across 3000 structured RNA motifs and uncovered pronounced binding preferences for G-quadruplexes and, notably, for a specific internal loop motif containing a U-U mismatch (K _Dapp = 0.31 µM). Furthermore, a motif extraction strategy was used to enable detailed structural investigation. The X-ray crystal structure of the amiloride-RNA complex provides the first structural evidence that amiloride recognizes a U residue within a naturally occurring RNA context via its signature complementary hydrogen bonding interactions.