{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,14]],"date-time":"2026-10-14T08:49:51Z","timestamp":1791967791293,"version":"4.3.4"},"reference-count":51,"publisher":"Oxford University Press (OUP)","issue":"19","license":[{"start":{"date-parts":[[2026,10,14]],"date-time":"2026-10-14T00:00:00Z","timestamp":1791936000000},"content-version":"vor","delay-in-days":1,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001711","name":"Swiss National Science Foundation","doi-asserted-by":"publisher","award":["200021_182056"],"award-info":[{"award-number":["200021_182056"]}],"id":[{"id":"10.13039\/501100001711","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,10,13]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>The number of internal repeat modules remains a major limiting factor in the construction and functional exploration of long repeat proteins. As a solution, we present High-Utility Recursive Directional Ligation for Extended Repeat proteins (HURDLER)\u2014a modular cloning strategy combining codon optimization, seamless insertion of restriction sites, and modified recursive directional ligation. It presents a very robust technique to clone DNA for large repeat proteins, which may also contain non-repetitive parts, with high fidelity requiring a minimal number of individual cloning steps. Using HURDLER, we cloned designed armadillo repeat proteins with up to 85 internal modules, corresponding to &amp;gt;3700 residues. The method also generates intermediate variants with defined numbers of internal repeats, enabling systematic investigation of how the number of modules influences protein structure and function. HURDLER thereby provides a reliable solution to overcome restrictions in the overall number of internal modules used in the construction of very long repeat proteins. Such proteins are increasingly utilized as building blocks in macromolecular protein assemblies with defined structures.<\/jats:p>","DOI":"10.1093\/nar\/gkag988","type":"journal-article","created":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T11:51:25Z","timestamp":1790855485000},"source":"Crossref","is-referenced-by-count":0,"title":["HURDLER: a cloning method to overcome length limits in designed repeat proteins"],"prefix":"10.1093","volume":"54","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7561-8727","authenticated-orcid":false,"given":"Wenzhao","family":"Dai","sequence":"first","affiliation":[{"name":"Department of Chemistry, University of Zurich , Winterthurerstrasse 190, 8057 Zurich ,","place":["Switzerland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3008-3957","authenticated-orcid":false,"given":"Matthias","family":"Schuster","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Zurich , Winterthurerstrasse 190, 8057 Zurich ,","place":["Switzerland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0475-438X","authenticated-orcid":false,"given":"Oliver","family":"Zerbe","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Zurich , Winterthurerstrasse 190, 8057 Zurich ,","place":["Switzerland"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2026,10,14]]},"reference":[{"key":"2026101404153825100_B1","doi-asserted-by":"crossref","first-page":"a023812","DOI":"10.1101\/cshperspect.a023812","article-title":"Synthetic DNA synthesis and assembly: putting the synthetic in synthetic biology","volume":"9","author":"Hughes","year":"2017","journal-title":"Cold Spring Harb Perspect Biol"},{"key":"2026101404153825100_B2","doi-asserted-by":"publisher","first-page":"8281","DOI":"10.1021\/ma020892i","article-title":"A new cloning method for the preparation of long repetitive polypeptides without a sequence requirement","volume":"35","author":"Won","year":"2002","journal-title":"Macromolecules"},{"key":"2026101404153825100_B3","doi-asserted-by":"publisher","first-page":"3551","DOI":"10.1021\/acssynbio.2c00355","article-title":"A user\u2019s guide to Golden Gate cloning methods and standards","volume":"11","author":"Bird","year":"2022","journal-title":"ACS Synth Biol"},{"key":"2026101404153825100_B4","doi-asserted-by":"publisher","first-page":"343","DOI":"10.1038\/nmeth.1318","article-title":"Enzymatic assembly of DNA molecules up to several hundred kilobases","volume":"6","author":"Gibson","year":"2009","journal-title":"Nat Methods"},{"key":"2026101404153825100_B5","doi-asserted-by":"publisher","first-page":"357","DOI":"10.1021\/bm015630n","article-title":"Genetically encoded synthesis of protein-based polymers with precisely specified molecular weight and sequence by recursive directional ligation: examples from the elastin-like polypeptide system","volume":"3","author":"DE","year":"2002","journal-title":"Biomacromolecules"},{"key":"2026101404153825100_B6","doi-asserted-by":"publisher","first-page":"251","DOI":"10.1038\/s41586-019-1361-6","article-title":"Controlling protein assembly on inorganic crystals through designed protein interfaces","volume":"571","author":"Pyles","year":"2019","journal-title":"Nature"},{"key":"2026101404153825100_B7","doi-asserted-by":"publisher","first-page":"651","DOI":"10.1111\/1751-7915.12081","article-title":"Recombinant DNA production of spider silk proteins","volume":"6","author":"Tokareva","year":"2013","journal-title":"Microb Biotechnol"},{"key":"2026101404153825100_B8","doi-asserted-by":"publisher","first-page":"1282","DOI":"10.1016\/j.jmb.2007.12.014","article-title":"Designed armadillo repeat proteins as general peptide-binding scaffolds: consensus design and computational optimization of the hydrophobic core","volume":"376","author":"Parmeggiani","year":"2008","journal-title":"J Mol Biol"},{"key":"2026101404153825100_B9","doi-asserted-by":"publisher","first-page":"D629","DOI":"10.1093\/nar\/gkac975","article-title":"REBASE: a database for DNA restriction and modification: enzymes, genes and genomes","volume":"51","author":"Roberts","year":"2023","journal-title":"Nucleic Acids Res"},{"key":"2026101404153825100_B10","doi-asserted-by":"publisher","first-page":"751","DOI":"10.1038\/90802","article-title":"Cell-free translation reconstituted with purified components","volume":"19","author":"Shimizu","year":"2001","journal-title":"Nat Biotechnol"},{"key":"2026101404153825100_B11","doi-asserted-by":"publisher","first-page":"eadg3683","DOI":"10.1126\/sciadv.adg3683","article-title":"Peptidomimetic antibiotics disrupt the lipopolysaccharide transport bridge of drug-resistant Enterobacteriaceae","volume":"9","author":"Schuster","year":"2023","journal-title":"Sci Adv"},{"key":"2026101404153825100_B12","doi-asserted-by":"publisher","first-page":"38","DOI":"10.1016\/j.jsb.2006.05.009","article-title":"EMAN2: an extensible image processing suite for electron microscopy","volume":"157","author":"Tang","year":"2007","journal-title":"J Struct Biol"},{"key":"2026101404153825100_B13","doi-asserted-by":"publisher","first-page":"290","DOI":"10.1038\/nmeth.4169","article-title":"cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination","volume":"14","author":"Punjani","year":"2017","journal-title":"Nat Methods"},{"key":"2026101404153825100_B14","doi-asserted-by":"publisher","first-page":"292","DOI":"10.1093\/nar\/28.1.292","article-title":"Codon usage tabulated from international DNA sequence databases: status for the year 2000","volume":"28","author":"Nakamura","year":"2000","journal-title":"Nucleic Acids Res"},{"key":"2026101404153825100_B15","doi-asserted-by":"publisher","first-page":"D452","DOI":"10.1093\/nar\/gkaa1097","article-title":"RepeatsDB in 2021: improved data and extended classification for protein tandem repeat structures","volume":"49","author":"Paladin","year":"2021","journal-title":"Nucleic Acids Res"},{"key":"2026101404153825100_B16","doi-asserted-by":"publisher","first-page":"580","DOI":"10.1038\/nature16162","article-title":"Exploring the repeat protein universe through computational protein design","volume":"528","author":"Brunette","year":"2015","journal-title":"Nature"},{"key":"2026101404153825100_B17","doi-asserted-by":"publisher","first-page":"3526","DOI":"10.1021\/jacs.6b00099","article-title":"Structure and energetic contributions of a designed modular peptide-binding protein with picomolar affinity","volume":"138","author":"Hansen","year":"2016","journal-title":"J Am Chem Soc"},{"key":"2026101404153825100_B18","doi-asserted-by":"publisher","first-page":"1572","DOI":"10.1002\/pro.2535","article-title":"Crystal structures of designed armadillo repeat proteins: implications of construct design and crystallization conditions on overall structure","volume":"23","author":"Reichen","year":"2014","journal-title":"Protein Sci"},{"key":"2026101404153825100_B19","doi-asserted-by":"publisher","first-page":"497","DOI":"10.1016\/S0969-2126(03)00076-5","article-title":"Design of stable alpha-helical arrays from an idealized TPR motif","volume":"11","author":"Main","year":"2003","journal-title":"Structure"},{"key":"2026101404153825100_B20","doi-asserted-by":"publisher","first-page":"16029","DOI":"10.1073\/pnas.252537899","article-title":"Consensus-derived structural determinants of the ankyrin repeat motif","volume":"99","author":"Mosavi","year":"2002","journal-title":"Proc Natl Acad Sci USA"},{"key":"2026101404153825100_B21","doi-asserted-by":"publisher","first-page":"318","DOI":"10.1021\/acs.biochem.2c00083","article-title":"Improved repeat protein stability by combined consensus and computational protein design","volume":"62","author":"Michel","year":"2023","journal-title":"Biochemistry"},{"key":"2026101404153825100_B22","doi-asserted-by":"publisher","first-page":"167","DOI":"10.1038\/nsmb.2938","article-title":"Control of repeat-protein curvature by computational protein design","volume":"22","author":"Park","year":"2015","journal-title":"Nat Struct Mol Biol"},{"key":"2026101404153825100_B23","doi-asserted-by":"publisher","first-page":"e2514871122","DOI":"10.1073\/pnas.2514871122","article-title":"Inhibition of ice recrystallization with designed twistless helical repeat proteins","volume":"122","author":"de\u00a0Haas","year":"2025","journal-title":"Proc Natl Acad Sci USA"},{"key":"2026101404153825100_B24","doi-asserted-by":"publisher","first-page":"800","DOI":"10.1107\/S0907444907024353","article-title":"Structure and stability of designed TPR protein superhelices: unusual crystal packing and implications for natural TPR proteins","volume":"63","author":"Kajander","year":"2007","journal-title":"Acta Crystallogr D Biol Crystallogr"},{"key":"2026101404153825100_B25","doi-asserted-by":"publisher","first-page":"1058","DOI":"10.1111\/j.1742-4658.2009.07549.x","article-title":"Crystal structure of a designed tetratricopeptide repeat module in complex with its peptide ligand","volume":"277","author":"Cortajarena","year":"2010","journal-title":"FEBS J"},{"key":"2026101404153825100_B26","doi-asserted-by":"publisher","first-page":"898","DOI":"10.1038\/s41586-024-07188-4","article-title":"Blueprinting extendable nanomaterials with standardized protein blocks","volume":"627","author":"Huddy","year":"2024","journal-title":"Nature"},{"key":"2026101404153825100_B27","doi-asserted-by":"publisher","first-page":"1015","DOI":"10.1002\/pro.2085","article-title":"Structure-based optimization of designed armadillo-repeat proteins","volume":"21","author":"Madhurantakam","year":"2012","journal-title":"Protein Sci"},{"key":"2026101404153825100_B28","doi-asserted-by":"publisher","first-page":"353","DOI":"10.1038\/nchem.2673","article-title":"Computational design of self-assembling cyclic protein homo-oligomers","volume":"9","author":"Fallas","year":"2017","journal-title":"Nature Chem"},{"key":"2026101404153825100_B29","doi-asserted-by":"publisher","first-page":"1942","DOI":"10.1002\/pro.3229","article-title":"Structures of designed armadillo repeat proteins binding to peptides fused to globular domains","volume":"26","author":"Hansen","year":"2017","journal-title":"Protein Sci"},{"key":"2026101404153825100_B30","doi-asserted-by":"publisher","first-page":"4467","DOI":"10.1016\/j.jmb.2016.09.012","article-title":"Computationally designed armadillo repeat proteins for modular peptide recognition","volume":"428","author":"Reichen","year":"2016","journal-title":"J Mol Biol"},{"key":"2026101404153825100_B31","doi-asserted-by":"publisher","first-page":"147","DOI":"10.1016\/j.jsb.2013.07.012","article-title":"Modular peptide binding: from a comparison of natural binders to designed armadillo repeat proteins","volume":"185","author":"Reichen","year":"2014","journal-title":"J Struct Biol"},{"key":"2026101404153825100_B32","doi-asserted-by":"publisher","first-page":"7823","DOI":"10.1038\/s41467-023-43596-2","article-title":"Unexpected dynamics in femtomolar complexes of binding proteins with peptides","volume":"14","author":"Cucuzza","year":"2023","journal-title":"Nat Commun"},{"key":"2026101404153825100_B33","doi-asserted-by":"publisher","first-page":"240","DOI":"10.1021\/sb300028q","article-title":"A basis set of de novo coiled-coil peptide oligomers for rational protein design and synthetic biology","volume":"1","author":"Fletcher","year":"2012","journal-title":"ACS Synth Biol"},{"key":"2026101404153825100_B34","doi-asserted-by":"publisher","first-page":"440","DOI":"10.1002\/(SICI)1097-0134(19990601)35:4&lt;440::AID-PROT7&gt;3.0.CO;2-Y","article-title":"A fast algorithm for genome-wide analysis of proteins with repeated sequences","volume":"35","author":"Pellegrini","year":"1999","journal-title":"Proteins"},{"key":"2026101404153825100_B35","doi-asserted-by":"publisher","first-page":"10","DOI":"10.1038\/s42004-025-01817-3","article-title":"Designing novel solenoid proteins with in silico evolution","volume":"9","author":"Pretorius","year":"2025","journal-title":"Commun Chem"},{"key":"2026101404153825100_B36","doi-asserted-by":"publisher","first-page":"4508","DOI":"10.1093\/bioinformatics\/btaa558","article-title":"DNA Chisel, a versatile sequence optimizer","volume":"36","author":"Zulkower","year":"2020","journal-title":"Bioinformatics"},{"key":"2026101404153825100_B37","doi-asserted-by":"publisher","first-page":"563","DOI":"10.1016\/j.jmb.2014.11.005","article-title":"A general computational approach for repeat protein design","volume":"427","author":"Parmeggiani","year":"2015","journal-title":"J Mol Biol"},{"key":"2026101404153825100_B38","doi-asserted-by":"publisher","first-page":"208","DOI":"10.1016\/j.sbi.2021.02.002","article-title":"Repeat proteins: designing new shapes and functions for solenoid folds","volume":"68","author":"Gidley","year":"2021","journal-title":"Curr Opin Struct Biol"},{"key":"2026101404153825100_B39","doi-asserted-by":"publisher","first-page":"585","DOI":"10.1038\/nature16191","article-title":"Rational design of alpha-helical tandem repeat proteins with closed architectures","volume":"528","author":"Doyle","year":"2015","journal-title":"Nature"},{"key":"2026101404153825100_B40","doi-asserted-by":"publisher","first-page":"29","DOI":"10.1038\/nchembio.1966","article-title":"De novo design of a four-fold symmetric TIM-barrel protein with atomic-level accuracy","volume":"12","author":"Huang","year":"2016","journal-title":"Nat Chem Biol"},{"key":"2026101404153825100_B41","doi-asserted-by":"publisher","first-page":"2294","DOI":"10.1038\/s41467-021-22276-z","article-title":"Design of multi-scale protein complexes by hierarchical building block fusion","volume":"12","author":"Hsia","year":"2021","journal-title":"Nat Commun"},{"key":"2026101404153825100_B42","doi-asserted-by":"publisher","article-title":"Nucleation limited assembly and polarized growth of a de novo-designed allosterically modulatable protein filament","author":"Shen","DOI":"10.1101\/2024.09.20.613980"},{"key":"2026101404153825100_B43","doi-asserted-by":"publisher","first-page":"1272","DOI":"10.1016\/j.jmb.2016.01.017","article-title":"Generation of fluorogen-activating designed ankyrin repeat proteins (FADAs) as versatile sensor tools","volume":"428","author":"Sch\u00fctz","year":"2016","journal-title":"J Mol Biol"},{"key":"2026101404153825100_B44","doi-asserted-by":"publisher","first-page":"8191","DOI":"10.1038\/s41467-023-43608-1","article-title":"Directing polymorph specific calcium carbonate formation with de novo protein templates","volume":"14","author":"Davila-Hernandez","year":"2023","journal-title":"Nat Commun"},{"key":"2026101404153825100_B45","doi-asserted-by":"publisher","article-title":"Controlling semiconductor growth with structured de novo protein interfaces","author":"Saragovi","DOI":"10.1101\/2024.06.24.600095"},{"key":"2026101404153825100_B46","doi-asserted-by":"publisher","first-page":"547","DOI":"10.1038\/s41586-021-04184-w","article-title":"De novo protein design by deep network hallucination","volume":"600","author":"Anishchenko","year":"2021","journal-title":"Nature"},{"key":"2026101404153825100_B47","doi-asserted-by":"publisher","first-page":"581","DOI":"10.1038\/s41586-023-05909-9","article-title":"De novo design of modular peptide-binding proteins by superhelical matching","volume":"616","author":"Wu","year":"2023","journal-title":"Nature"},{"key":"2026101404153825100_B48","doi-asserted-by":"publisher","first-page":"100564","DOI":"10.1016\/j.crmeth.2023.100564","article-title":"An efficient cloning method to expand vector and restriction site compatibility of Golden Gate Assembly","volume":"3","author":"Sorida","year":"2023","journal-title":"Cell Reports Methods"},{"key":"2026101404153825100_B49","doi-asserted-by":"publisher","first-page":"D535","DOI":"10.1093\/nar\/gkz915","article-title":"CRISPRCasdb a successor of CRISPRdb containing CRISPR arrays and cas genes from complete genome sequences, and tools to download and query lists of repeats and spacers","volume":"48","author":"Pourcel","year":"2020","journal-title":"Nucleic Acids Res"},{"key":"2026101404153825100_B50","doi-asserted-by":"publisher","first-page":"D192","DOI":"10.1093\/nar\/gkaa1047","article-title":"Rfam 14: expanded coverage of metagenomic, viral and microRNA families","volume":"49","author":"Kalvari","year":"2021","journal-title":"Nucleic Acids Res"},{"key":"2026101404153825100_B51","doi-asserted-by":"publisher","first-page":"D264","DOI":"10.1093\/nar\/gkaf1016","article-title":"Ribocentre-aptamer: an integrative, structure-focused database for RNA aptamers","volume":"54","author":"Lu","year":"2026","journal-title":"Nucleic Acids Res"}],"container-title":["Nucleic Acids Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/academic.oup.com\/nar\/article-pdf\/54\/19\/gkag988\/71926858\/gkag988.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/nar\/article-pdf\/54\/19\/gkag988\/71926858\/gkag988.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,14]],"date-time":"2026-10-14T08:15:46Z","timestamp":1791965746000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/nar\/article\/doi\/10.1093\/nar\/gkag988\/8915704"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,10,13]]},"references-count":51,"journal-issue":{"issue":"19","published-print":{"date-parts":[[2026,10,13]]}},"URL":"https:\/\/doi.org\/10.1093\/nar\/gkag988","relation":{},"ISSN":["0305-1048","1362-4962"],"issn-type":[{"value":"0305-1048","type":"print"},{"value":"1362-4962","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2026,10,28]]},"published":{"date-parts":[[2026,10,13]]},"article-number":"gkag988"}}