A tunable orthogonal coiled-coil interaction toolbox for engineering mammalian cells

ABSTRACT Protein interactions guide most cellular processes. Orthogonal hetero-specific protein–protein interaction domains may facilitate better control of engineered biological systems.

Here, we report a tunable de novo designed set of orthogonal coiled-coil (CC) peptide heterodimers (called the NICP set) and its application for the regulation of diverse cellular processes,

from cellular localization to transcriptional regulation. We demonstrate the application of CC pairs for multiplex localization in single cells and exploit the interaction strength and

variable stoichiometry of CC peptides for tuning of gene transcription strength. A concatenated CC peptide tag (CCC-tag) was used to construct highly potent CRISPR–dCas9-based

transcriptional activators and to amplify the response of light and small molecule-inducible transcription in cell culture as well as in vivo. The NICP set and its implementations represent

a valuable toolbox of minimally disruptive modules for the recruitment of versatile functional domains and regulation of cellular processes for synthetic biology. Access through your

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BACTERIAL TWO-HYBRID FOR RAPID CHARACTERIZATION OF PROTEIN–PROTEIN INTERACTIONS AND ITERATIVE PROTEIN DESIGN Article Open access 02 August 2023 DESIGNER PROTEIN ASSEMBLIES WITH TUNABLE PHASE

DIAGRAMS IN LIVING CELLS Article 13 July 2020 PROGRAMMABLE DE NOVO DESIGNED COILED COIL-MEDIATED PHASE SEPARATION IN MAMMALIAN CELLS Article Open access 02 December 2023 DATA AVAILABILITY

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by grants from the Slovenian Research Agency (nos. P4-0176, J1-9173, J3-7034 and N4-0080), ERC grant MaCChines to R.J, Horizon2020 CSA Bioroboost and ERANET project MediSurf. T.L. is

partially supported by the UNESCO-L’OREAL national fellowship ‘For Women in Science’. We thank H. Gradišar for providing the sequences of CC peptides and for valuable advice. AUTHOR

INFORMATION AUTHORS AND AFFILIATIONS * Department of Synthetic Biology and Immunology, National Institute of Chemistry, Ljubljana, Slovenia Tina Lebar, Duško Lainšček, Estera Merljak, Jana

Aupič & Roman Jerala Authors * Tina Lebar View author publications You can also search for this author inPubMed Google Scholar * Duško Lainšček View author publications You can also

search for this author inPubMed Google Scholar * Estera Merljak View author publications You can also search for this author inPubMed Google Scholar * Jana Aupič View author publications You

can also search for this author inPubMed Google Scholar * Roman Jerala View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS T.L. and E.M.

prepared the plasmid constructs and performed the experiments on cell culture. D.L. performed the experiments on mice. J.A. performed the bioinformatics analysis. T.L. and R.J. designed and

analyzed the experiments and wrote the manuscript. R.J. conceived the study. CORRESPONDING AUTHOR Correspondence to Roman Jerala. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare

no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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ARTICLE Lebar, T., Lainšček, D., Merljak, E. _et al._ A tunable orthogonal coiled-coil interaction toolbox for engineering mammalian cells. _Nat Chem Biol_ 16, 513–519 (2020).

https://doi.org/10.1038/s41589-019-0443-y Download citation * Received: 11 March 2019 * Revised: 21 October 2019 * Accepted: 25 November 2019 * Published: 06 January 2020 * Issue Date: May

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