De novo designed peptides for cellular delivery and subcellular localisation
Open accessAbstract
Increasingly, it is possible to design peptide and protein assemblies de novo from first principles or computationally. This approach provides new routes to functional synthetic polypeptides, including designs to target and bind proteins of interest. Much of this work has been developed in vitro. Therefore, a challenge is to deliver de novo polypeptides efficiently to sites of action within cells. Here we describe the design, characterisation, intracellular delivery, and subcellular localisation of a de novo synthetic peptide system. This system comprises a dual-function basic peptide, programmed both for cell penetration and target binding, and a complementary acidic peptide that can be fused to proteins of interest and introduced into cells using synthetic DNA. The designs are characterised in vitro using biophysical methods and X-ray crystallography. The utility of the system for delivery into mammalian cells and subcellular targeting is demonstrated by marking organelles and actively engaging functional protein complexes.
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The Asn17-Asn17 hydrogen bond in CC-Di and the Asp@e-Arg@a hydrogen bonds/salt bridges in apCC-Di determine the formation of parallel and antiparallel dimers, respectively.
“Of notable importance are the Asn17-Asn17 hydrogen bond in CC-Di, and the Asp@e-Arg@a hydrogen bonds/salt bridges in apCC-Di, which determine the formation of parallel and antiparallel dimers, respectively.33,89”
From Assessment of the Topology and Oligomerisation States of Coiled Coils Using Metadynamics with Conformational Restraints (2025) · cites this paper as [89]
apCC-Di experimentally only accesses the antiparallel dimer state, also has a lower ΔGbind,total°/helix (−5.3 ± 0.5 kcal mol-1) compared to the other single-state coiled coils, but it has a lower melting point.
“We note that apCC-Di, which experimentally only accesses the antiparallel dimer state, also has a lower ΔGbind,total◦/helix (−5.3 ± 0.5 kcal mol–1) compared to the other single-state coiled coils, but it has a lower melting point.89”
From Assessment of the Topology and Oligomerisation States of Coiled Coils Using Metadynamics with Conformational Restraints (2025) · cites this paper as [89]
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“Many engineering efforts have used peptides to permeate membranes by various mechanisms and increase cytosolic delivery of cargo (Akishiba et al., 2017; Chen et al., 2021; Rhys et al., 2022; Sakamoto et al., 2021).”
BackgroundGrafted Coiled-Coil Peptides as Multivalent Scaffolds for Protein Recognition
Acevedo‐Jake, A. M., Mylemans, B., Kay, D. F., Zhang, P., Korona, B., Rhys, G. G., Leney, A. C., Huang, D. T. C., Edwards, T. A., Itzhaki, L. S., et al. (2025) · ACS Chemical Biology · doi:10.1021/acschembio.5c00137 · Cited by 9 · see citing works
Self-citationDe novo design of triosephosphate isomerases using generative language modelsPreprint
Romero‐Romero, S., Braun, A. E., Kossendey, T., Ferruz, N., Schmidt, S., & Höcker, B. (2024) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2024.11.10.622869 · Cited by 11 · see citing works
Self-citationFerrer, P. R., & Sakiyama‐Elbert, S. E. (2024) · Journal of Neural Engineering · doi:10.1088/1741-2552/ad680a · Cited by 6 · see citing works
“De novo proteins can also be designed to change conformation in response to specific stimulus, possess reduced immunogenicity and modified to potentially cross the BBB and BSCB [63, 64].”
Menke, F. S., Wicher, B., Maurizot, V., & Huc, I. (2023) · Angewandte Chemie · doi:10.1002/ange.202217325 · Cited by 6 · see citing works
Hakata, Y., Yamashita, K., Hashimoto, S., Ohtsuki, T., Miyazawa, M., & Kitamatsu, M. (2023) · Pharmaceutics · doi:10.3390/pharmaceutics15041048 · Cited by 5 · see citing works
De novo designed 3-helix bundle peptides and proteins with controlled topology and stability
Leng, X., Albanese, K. I., Golub, L. R., Norman, A. A., Clayden, J., & Woolfson, D. N. (2025) · Chemical Science · doi:10.1039/d5sc05576h · Cited by 6 · see citing works
Self-citationExchange, promiscuity, and orthogonality in de novo designed coiled-coil peptide assemblies
Kurgan, K. W., Martin, F. J. O., Dawson, W., Brunnock, T., Orr‐Ewing, A. J., & Woolfson, D. N. (2024) · Chemical Science · doi:10.1039/d4sc06329e · Cited by 5 · see citing works
Self-citationDe Novo Design of Parallel and Antiparallel A3B3 Heterohexameric α-Helical Barrels
Chubb, J. J., Albanese, K. I., Rodger, A., & Woolfson, D. N. (2025) · Biochemistry · doi:10.1021/acs.biochem.4c00584 · Cited by 4 · see citing works
“Therefore, we used the proximity-based fluorescence quenching assay illustrated in Figure 2G. 10,37,43 The quencher selenomethionine (MSe, Ω ) was incorporated at the C -terminal g site of CC-Hex2-A-3-c , and the fluorescent 4-cyanophenylalanine (4CF, Δ ) was placed at either the N -terminal c site…”
Self-citationDirectional Engineering of Cyclic β-Hairpin Peptides for PET/CT Imaging of PD-L1
Liu, C., Zhang, S., Chen, J., Geng, H. R., Li, H., Zhang, Z., Lu, Q., Xu, L., Li, J., Zhang, H., et al. (2026) · Journal of the American Chemical Society · doi:10.1021/jacs.5c21595 · Cited by 4 · see citing works
Design and Selection of Heterodimerizing Helical Hairpins for Synthetic Biology
Smith, A. J., Naudin, E. A., Edgell, C. L., Baker, E. G., Mylemans, B., FitzPatrick, L., Herman, A., Rice, H. M., Andrews, D. M., Tigue, N. J., et al. (2023) · ACS Synthetic Biology · doi:10.1021/acssynbio.3c00231 · Cited by 4 · see citing works
Self-citationNotari, E., Wood, C. W., & Michel, J. (2025) · Journal of Chemical Theory and Computation · doi:10.1021/acs.jctc.4c01695 · Cited by 4 · see citing works
Beck, J., & Romero‐Romero, S. (2026) · Biochemical Society Transactions · doi:10.1042/bst20253060 · Cited by 2 · see citing works
Zhou, S., Nishimura, F., Wada, K., Fujii, K., Kondo, T., Watanabe, K., Imai, Y., Ohtsuki, T., & Kitamatsu, M. (2024) · Bioorganic & Medicinal Chemistry Letters · doi:10.1016/j.bmcl.2024.129915 · Cited by 1 · see citing works
Controlling cell architecture with protein design
Cross, J. A., Woolfson, D. N., & Dodding, M. P. (2025) · Current Opinion in Cell Biology · doi:10.1016/j.ceb.2025.102569 · Cited by 1 · see citing works
Self-citationHu, Z., Jiang, Y., Hou, K., Miao, D., Ye, Y., Huang, C., & Xiong, R. (2026) · Chemical Engineering Journal · doi:10.1016/j.cej.2026.175687 · Cited by 1 · see citing works
Enzyme-Free Phosphorylation with Kinetic Gating in a De Novo Coiled-Coil System
Poprawa, S., Hoja, N., Hipp, C., King, R. O., Soria‐Carrera, H., Woolfson, D. N., Sattler, M., Kaila, V. R. I., & Boekhoven, J. (2026) · Journal of the American Chemical Society · doi:10.1021/jacs.6c02162 · Cited by 1 · see citing works
Self-citationDe novo grafted coiled-coil peptides as p53/ h DM2 inhibitors
Spain, F., Giménez, D., Acevedo‐Jake, A. M., Mylemans, B., Brooks, N. J., Korona, B., Huang, D. T. C., Edwards, T. A., Leney, A. C., Itzhaki, L. S., et al. (2026) · RSC Chemical Biology · doi:10.1039/d6cb00063k · Cited by 1 · see citing works
Self-citationProtein Modifications for Cellular Protein Delivery
Arafiles, J. V. V., Franke, J., Dubatouka, P., Franz, L., & Hackenberger, C. P. R. (2026) · Chemical Reviews · doi:10.1021/acs.chemrev.6c00267 · Cited by 1 · see citing works
“342,343 We expect continued progress in this area through further advances in our understanding of translocation mechanisms and the physicochemical parameters that influence delivery.”
Rationally seeded computational protein designPreprint
Albanese, K. I., Petrenas, R., Pirro, F., Naudin, E. A., Borucu, U., Dawson, W., Scott, D. A., Leggett, G. J., Weiner, O. D., Oliver, T. A. A., et al. (2023) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2023.08.25.554789 · Cited by 1 · see citing works
Self-citationDe novo designed bifunctional proteins for targeted protein degradationPreprint
Mylemans, B., Korona, B., Acevedo‐Jake, A. M., MacRae, A., Edwards, T. A., Huang, D. T. C., Wilson, A. J., Itzhaki, L. S., & Woolfson, D. N. (2025) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.64898/2025.12.22.695915 · Cited by 2 · see citing works
Self-citationCoiled-coil scaffolds for Targeted Protein DegradationPreprint
Korona, B., Mylemans, B., Acevedo‐Jake, A. M., Huang, D. T., Edwards, T. A., Wilson, A. J., Woolfson, D. N., & Itzhaki, L. S. (2025) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.64898/2025.12.22.695764 · Cited by 1 · see citing works
“…by poly(GS) spacers (Table S1): (i) the BAD peptide as the BCL-x L binding motifs; (ii) two different degrons (UBR and ODC); and (iii) four different de novo designed 30-residue coiled-coil modules (anti-parallel dimer, parallel trimer, parallel and anti-parallel tetramer) 28, 47, 48, 54, 57, 58 .”
Self-citationAlbanese, K. I., Barbe, S., Tagami, S., Woolfson, D. N., & Schiex, T. (2025) · Nature Reviews Methods Primers · doi:10.1038/s43586-025-00383-1 · Cited by 96 · see citing works
“…350–352 , antibodies and immunotherapies 65,275,276,353–360 , biosensors 311,361–365 , allosteric modulators 61,62 , cellular delivery agents 366 , nanomaterials 38,60,367,368 , ion transporters and nanopores 369–372 , protein machines 373 , protein binders 28,259,374 and enzymes with new…”
Self-citationExchange, promiscuity, and orthogonality in de novo designed coiled-coil peptide assembliesPreprint
Kurgan, K. W., Martin, F. J. O., Dawson, W., Brunnock, T., Orr‐Ewing, A. J., & Woolfson, D. N. (2024) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2024.09.01.610678
“2,4,10 De 14-16 Recent in-cell studies have highlighted the potential use of de novo coiled-coil design for targeting natural protein-protein interactions 17 and effecting allosteric activation of these.”
BackgroundSelf-citationDe novo design of parallel and antiparallel A 3 B 3 heterohexameric α-helical barrelsPreprint
Chubb, J. J., Albanese, K. I., Rodger, A., & Woolfson, D. N. (2024) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2024.09.25.614870
Self-citationNotari, E., Wood, C. W., & Michel, J. (2024) · ChemRxiv · doi:10.26434/chemrxiv-2024-t02df
Computational Design of Therapeutic Peptides
Choudhury, P. R., Yadav, S., Jaiswal, S., Khanna, T., & Mathur, P. (2025) · BENTHAM SCIENCE PUBLISHERS eBooks · doi:10.2174/9798898812164125080005
Chen, C., Du, X., Yang, J., Zeng, Y., Xie, H., Wang, D., Qi, P., & Zeng, Y. (2026) · Frontiers in Pharmacology · doi:10.3389/fphar.2026.1796070
“…defect in the epilepsy model, AI can guide the design of smart nanoparticles modified with a specific presynaptic membrane targeting peptide and capable of releasing GABA reuptake inhibitors in response to changes in the local potassium ion concentration (Rhys et al., 2022; Naskar et al., 2021).”
De Novo-Designed Bifunctional Proteins for Targeted Protein Degradation
Mylemans, B., Korona, B., Acevedo‐Jake, A. M., MacRae, A., Edwards, T. A., Huang, D. T., Wilson, A. J., Itzhaki, L. S., & Woolfson, D. N. (2026) · Journal of the American Chemical Society · doi:10.1021/jacs.6c07593 · Cited by 1 · see citing works
Self-citationLiang, Q., Wang, S., Sun, N., Zhu, C., Liu, Z., & Mou, H. (2026) · Foods · doi:10.3390/foods15203617
- Authors
- Rhys, G. G., Cross, J. A., Dawson, W. M., Thompson, H. F., Shanmugaratnam, S., Savery, N. J., Dodding, M. P., Höcker, B., & Woolfson, D. N.
- Affiliations
- University of Bayreuth (DE)
- University of Bristol (GB)
- Corresponding author
- Höcker, Birte
- Woolfson, Derek N.
- ORCID
- Venue
- Nature Chemical Biology
- Published
- 2022-07-14
- Volume
- 18
- Issue
- 9
- Pages
- 999-1004
- ISSN
- 1552-4450 (ISSN-L)
- 1552-4469
- OpenAlex
- W4285386073
- Cited by
- 44
- References
- 73
- Citations by year
- 2026: 9 · 2025: 10 · 2024: 13 · 2023: 10 · 2022: 2
- FWCI
- 2.86
- Type
- journal article
- Language
- en
- Open access
- green
- orca.cardiff.ac.uk
- Locations
- Nature Chemical Biology (publishedVersion)
- PubMed (publishedVersion)
- ORCA Online Research @Cardiff (Cardiff University) (acceptedVersion)
- ERef Bayreuth (University of Bayreuth) (submittedVersion)
- HZB Repository (Helmholtz-Zentrum Berlin für Materialien und Energie GmbH (HZB)) (submittedVersion)
- ORCA Online Research @Cardiff (Cardiff University) (submittedVersion, cc-by)
- Explore Bristol Research (submittedVersion, other-oa)
- Funders
- European Commission
- University of Bristol (BB/L01386X/1, EP/L016494/1)
- Lister Institute of Preventive Medicine
- Engineering and Physical Sciences Research Council (EP/K03927X/1, EP/G036764/1, (BB/L01386X/1), EP/G036764, EP/L016494/1)
- Biotechnology and Biological Sciences Research Council (BB/L01386X/1, BB/W005581/1, BB/L01386X, EP /L016494/1, BB/S000917/1, L01386X)
- BrisSynBio (BB/L01386X/1, L01386X)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chemical Synthesis and Analysis
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Keywords
- de novo peptide design, intracellular delivery, subcellular localization, cell-penetrating peptides, synthetic polypeptides, protein fusion tags, acidic peptides, basic peptides, organelle targeting, synthetic DNA
- MeSH terms
- Animals, Mammals, Peptides, Organelles, Crystallography, X-Ray
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