Beyond Hybridisation: Biologically Meaningful Protein Interactions of Oligonucleotides

by Rob Kirby PhD, COO

Oligonucleotide therapeutics offer powerful ways to modulate disease biology, but achieving both efficacy and safety depends on a deep understanding of structure-activity relationships, not only for intended targets but also for unexpected off-target interactions. These themes are explored in a paper published in Nature Communications entitled ‘The nucleobase guanine at the 3′-terminus of oligonucleotide RGLS4326 drives off-target AMPAR inhibition and CNS toxicity'⁽¹⁾ ,which I was pleased to contribute as a co-author.

This study investigated why the first-generation anti-miR-17 oligonucleotide RGLS4326, developed for autosomal dominant polycystic kidney disease (ADPKD), was discontinued despite encouraging pharmacodynamic activity in a Phase 1b study. Chronic nonclinical toxicity studies revealed dose-limiting central nervous system toxicity, prompting further investigation into whether these effects were attributable to on-target miR-17 inhibition or to an alternative mechanism.

To support this effort, the team at Regulus Therapeutics engaged Metrion to apply its electrophysiology expertise to investigate potential off-target ion channel and receptor interactions that might explain the observed CNS toxicity.

The studies demonstrated that the CNS toxicity was not driven by miR-17 inhibition. Instead, the authors identified an unexpected , aptamer-like direct interaction between RGLS4326 and the AMPA receptor, a key mediator of excitatory neurotransmission in the brain. Through systematic structure–activity relationship analysis, the researchers identified a key structural determinant for this liability: a guanine nucleobase at the 3′ terminus of the oligonucleotide that enabled direct binding to the AMPA receptor.

With this mechanistic insight, a precise and minimal design change, replacing the 3′-terminal guanine with adenine, led to the discovery of the next-generation anti-miR-17 candidate, RGLS8429. This single-nucleotide substitution removed AMPA receptor interaction and associated CNS toxicity in the tested models while preserving potency against the intended miR-17 target.

More broadly, this work highlights an important lesson for the oligonucleotide field: oligonucleotides can, in some cases, engage in biologically meaningful off-target protein interactions, and safety liabilities may hinge on subtle structural features. Careful structure-activity relationship analysis, even at the level of a single nucleotide, can be decisive in enabling the progression of improved therapeutic candidates.

RGLS8429, the next generation compound described in the paper, is now progressing clinically as Farabursen. In 2025 Novartis acquired Regulus Therapeutics to advance Farabursen’s development for ADPKD.

1. Valencia, T., Yen, L.Y., Berman, C. et al.The nucleobase guanine at the 3’-terminus of oligonucleotide RGLS4326 drives off-target AMPAR inhibition and CNS toxicity. Nat Commun 16, 10762 (2025). https://www.nature.com/articles/s41467-025-65799-5