Transcriptome-Wide Studies of RNA-Targeted Small Molecules Provide a Simple and Selective r(CUG)^exp Degrader in Myotonic Dystrophy

Abstract

Myotonic dystrophy type 1 (DM1) is caused by a highly structured RNA repeat expansion, r(CUG) exp , harbored in the 3′ untranslated region (3′ UTR) of dystrophia myotonica protein kinase (DMPK) mRNA and drives disease through a gain-offunction mechanism. A panel of low-molecular-weight fragments capable of reacting with RNA upon UV irradiation was studied for cross-linking to r(CUG) exp in vitro, affording perimidin-2-amine diazirine (1) that bound to r(CUG) exp . The interactions between the small molecule and RNA were further studied by nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. Binding of 1 in DM1 myotubes was profiled transcriptome-wide, identifying 12 transcripts including DMPK that were bound by 1. Augmenting the functionality of 1 with cleaving capability created a chimeric degrader that specifically targets r(CUG) exp for elimination. The degrader broadly improved DM1-associated defects as assessed by RNA-seq, while having limited effects on healthy myotubes. This study (i) provides a platform to investigate molecular recognition of ligands directly in disease-affected cells; (ii) illustrates that RNA degraders can be more specific than the binders from which they are derived; and (iii) suggests that repeating transcripts can be selectively degraded due to the presence of multiple ligand binding sites.

Transcriptome-wide mapping of small-molecule RNA-binding sites in live cells

“…22 Conversely, the attachment point of the crosslink and enrichment handle of a hit compound from an FFF library screening, presents a convenient opportunity to create a modularly assembled small molecule that binds two sites simultaneously 23 or develop RNA degraders. 24 The background of pull-down observed in the vehicle (DMSO-treated) sample is typically ~1% and should not exceed 5%.…”

G-Quadruplex mRNAs Silencing with Inducible Ribonuclease Targeting Chimera for Precision Tumor Therapy

“…Compared to the target-binding strategy, the binding-and-degrading strategy of RIBOTAC offers unique advantages, as it turns functionally silent ligands to valuable RNA degraders with high catalytic turnovers . Pilot studies of RIBOTAC have demonstrated for degradation of different RNAs ranging from precursors of microRNA, − viral RNA, to mRNAs . However, small-molecule RNA ligands are still limited, and many disease-associated RNAs are untargetable.…”