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Synthetic biomolecular condensates enhance translation from a target mRNA in living cells

Nature Chemistry volume 17, pages 448–456 (2025)Cite this article

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Abstract

Biomolecular condensates composed of proteins and RNA are one approach by which cells regulate post-transcriptional gene expression. Their formation typically involves the phase separation of intrinsically disordered proteins with a target mRNA, sequestering the mRNA into a liquid condensate. This sequestration regulates gene expression by modulating translation or facilitating RNA processing. Here we engineer synthetic condensates using a fusion of an RNA-binding protein, the human Pumilio2 homology domain (Pum2), and a synthetic intrinsically disordered protein, an elastin-like polypeptide (ELP), that can bind and sequester a target mRNA transcript. In protocells, sequestration of a target mRNA largely limits its translation. Conversely, in Escherichia coli, sequestration of the same target mRNA increases its translation. We characterize the Pum2–ELP condensate system using microscopy, biophysical and biochemical assays, and RNA sequencing. This approach enables the modulation of cell function via the formation of synthetic biomolecular condensates that regulate the expression of a target protein.

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Fig. 1: The Pum2–ELP synthetic RNPG platform specifically binds mRNA tagged with a Pumilio2 recognition sequence.
Fig. 2: Synthetic RNPGs inhibit protein translation in protocells.
Fig. 3: Synthetic RNPGs enhance mRNA translation in live cells.
Fig. 4: Synthetic RNPGs are highly liquid biomolecular condensates.
Fig. 5: Quantification of mCherry protein abundance confirms synthetic RNPGs enhance protein translation and preferentially sequester target mRNA.
Fig. 6: Proposed model for translational enhancement by synthetic RNPGs.

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Data availability

The data that support the findings of this study are available within this Article and its Supplementary Information. Numerical and gel image source data have been provided as source files. RNA-seq data may be downloaded from the Gene Expression Omnibus accession number GSE277409. Image and material requests should be made to the corresponding author.

Code availability

The R script used to compute TPM is available with this Article as Supplementary Information.

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Acknowledgements

We thank the Duke Light Microscopy Core Facility for experimental support. A.C. acknowledges the support of the Air Force Office of Scientific Research (FA9550-20-1-0241), the National Institutes of Health (MIRA R35GM127042) and a ‘Beyond the Horizons’ award from the Pratt School of Engineering at Duke University. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Author notes

  1. These authors contributed equally: Daniel Mark Shapiro, Sonal Deshpande, Seyed Ali Eghtesadi.

Authors and Affiliations

  1. Department of Biomedical Engineering, Duke University, Durham, NC, USA

    Daniel Mark Shapiro, Sonal Deshpande, Seyed Ali Eghtesadi, Miranda Zhong, Cassio Mendes Fontes, David Fiflis, Dahlia Rohm, Junseon Min, Taranpreet Kaur, Joanna Peng, Max Ney, Jonathan Su, Yifan Dai, Aravind Asokan, Charles A. Gersbach & Ashutosh Chilkoti

  2. Department of Surgery, Duke University Medical Center, Durham, NC, USA

    David Fiflis, Aravind Asokan & Charles A. Gersbach

  3. Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO, USA

    Yifan Dai

  4. Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC, USA

    Dahlia Rohm & Aravind Asokan

  5. Center for Advanced Genomic Technologies, Duke University, Durham, NC, USA

    Charles A. Gersbach

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Contributions

D.M.S., S.D. and A.C. designed the experiments and wrote the manuscript. D.M.S. and S.D. collected all measurements, with exception of in vitro data for Supplementary Figs. 1–3 and 6 (collected by S.A.E.) and SPR data (collected by S.A.E., C.M.F. and J.M.). D.M.S. and S.D. analysed all data, with exception of SPR data (analysed by C.M.F.). D.M.S., S.A.E. and S.D. cloned plasmids and prepared cell lines. S.D. collected and analysed in vitro protocell measurements. D.M.S. collected and analysed all in vivo imaging data. D.M.S., M.Z. and M.N. collected and analysed photobleaching experiment data. T.K. analysed photobleaching recovery and loss rates. D.M.S., D.F. and A.A. developed and executed ultracentrifugation protocol to purify synthetic RNPGs. D.M.S., D.R. and C.A.G. executed and analysed RNA-seq and next-generation sequencing experiments. D.M.S., S.D., S.A.E. M.Z., J.S. and J.P. purified protein for characterization. D.M.S. and Y.D. performed and analysed in vivo growth assays. D.M.S., S.D. and A.C. wrote the manuscript. All the authors read and contributed revisions. All the authors contributed to discussions. A.C. supervised the work and acquired funding.

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Correspondence to Ashutosh Chilkoti.

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Extended material and methods, Supplementary Tables 1–5, Supplementary Figs. 1–16, Supplementary Notes 1–3 and references.

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Supplementary Code 1

Modified script to calculate TPM of RNA detected in RNA-seq.

Supplementary Table 1

Numerical source data for all plots in main text and selected supplementary figures.

Supplementary Table 6

Supplementary Table 7

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Shapiro, D.M., Deshpande, S., Eghtesadi, S.A. et al. Synthetic biomolecular condensates enhance translation from a target mRNA in living cells. Nat. Chem. 17, 448–456 (2025). https://doi.org/10.1038/s41557-024-01706-7

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