RNA-binding Landscape of Amiloride: Large-scale Profiling and Structural Basis of U–U Mismatch Recognition

Abstract

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 3,000 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.