Abstract
Targeting RNA with small molecules offers a strategy to modulate gene expression at undruggable targets. Traditional screens favor thermodynamically stable, low-entropy RNA motifs with defined conformations, yet these provide limited energetic leverage for functional modulation. Many RNAs instead sample dynamic structural ensembles that small molecules can repartition. Using group I self-splicing introns as a model, we identified the antineoplastic drug Mitoxantrone as a competitive inhibitor of RNA self-splicing (IC50 = 4.3 μM) that stabilizes the native conformation of the T4 td intron. Structure-activity analysis showed that the anthraquinone scaffold alone is insufficient, and basic amine-containing side chains are required for RNA structural modulation. Transcriptome-wide chemical probing in human cells revealed preferential binding to GC-rich structured regions, although only a subset showed structural change. Furthermore, global analysis of 5′ UTR ensembles showed altered structural heterogeneity and translation, demonstrating functional repartitioning of RNA conformational landscapes.
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Data availability
The data supporting the findings of this study are available from the corresponding authors upon request. Sequencing data have been deposited to the Gene Expression Omnibus (GEO) database, under the accession GSE302505. Raw MM files for analysis with DRACO are available from Zenodo (https://doi.org/10.5281/zenodo.15874381). Additional processed data are available at https://www.incarnatolab.com/datasets/Mitoxantrone_Zhang_2026.php. Source data for the figures and Supplementary Figure are provided as a Source Data file. Source data are provided with this paper.
Code availability
The source codes of DRACO v1.3, and of the diffShape utility are freely available from GitHub, under the GPLv3 license (https://github.com/dincarnato/draco and https://github.com/dincarnato/papers).
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Acknowledgements
This work was supported by a grant from the European Research Council (European Union’s Horizon Europe research and innovation program), grant agreement number 101124787 (RNAStrEnD) to D.I., and partly supported by the Intramural Research Program of the National Institutes of Health (NIH) (ZIABC011585). The contributions of the NIH author(s) were made as part of their official duties as NIH federal employees, are in compliance with agency policy requirements, and are considered Works of the United States Government. However, the findings and conclusions presented in this paper are those of the author(s) and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services.
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C.Z. and D.I. designed the experiments, with input from J.S.S., K.P., and M.D.W.; C.Z., I.B., T.I., M.L., and O.S.O. performed the experiments; R.B. performed the microscopy; M.L., O.S.O., M.D.W., and K.P. performed synthesis and characterization of the anthraquinone analogs; E.M. and D.I. optimized the DRACO algorithm and performed the bioinformatic analyses; M.C. supported the RiboLace analysis; C.Z., I.B., J.S.S., and D.I. wrote the article with input from all the authors; D.I. supervised the research.
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M.C. is the founder, director of, and a shareholder in IMMAGINA Biotechnology S.r.l. The remaining authors declare no competing interests.
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Zhang, C., Borovská, I., Iobashvili, T. et al. RNA functional modulation by Mitoxantrone via RNA structural ensemble repartitioning. Nat Commun (2026). https://doi.org/10.1038/s41467-026-70801-9
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DOI: https://doi.org/10.1038/s41467-026-70801-9
