{
  "abstract": "Peptide lasso-grafting strategies enable the transfer of established pharmacophores into compact protein scaffolds but often require optimization to preserve target binding and biological activity. Here, we report Random in-vitro Protein single-Loop Engineering (RiPLE), an mRNA-display strategy for the direct discovery of engineered ubiquitin binders, termed U-bodies, from a library containing a fully randomized surface loop of variable length. Using receptor tyrosine kinase-like orphan receptor 1 (ROR1) as a model target, we generated U-bodies through RiPLE and compared them with lasso-grafted U-bodies derived from a previously identified ROR1-binding macrocyclic peptide. Both approaches yielded U-bodies bearing single-digit nanomolar affinity to ROR1; however, RiPLE converged on de novo binding sequences that were distinct from the parental peptide. Notably, most selected U-bodies exhibited strong selectivity for ROR1 over the closely related receptor ROR2, whereas the parental peptide bound both receptors with high affinity. Representative ROR1-binding U-bodies also inhibited wound closure in ROR1-expressing MDA-MB-231 cells without detectable cytotoxicity. Applying RiPLE to the heterotrimeric G-protein subunit (GS) similarly yielded high-affinity binders and revealed sequence convergence patterns distinct from lasso-grafting. Together, these results establish RiPLE as a powerful and complementary alternative to peptide-guided lasso-grafting, enabling de novo evolution of functional binding surfaces directly within their final protein scaffold.",
  "abstract_shown": true,
  "affiliations": [
    "The University of Tokyo"
  ],
  "article_type": "New results",
  "arxiv": null,
  "author_total": 4,
  "authors": [
    {
      "affs": [
        0
      ],
      "name": "K. Colas"
    },
    {
      "name": "T. Ko"
    },
    {
      "name": "G. Philippe"
    },
    {
      "name": "H. Suga"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/b0d61vp4ghxb0.bib",
  "cited_by": 0,
  "doi": "10.64898/2026.09.21.753128",
  "fulltext_url": "https://www.biorxiv.org/content/10.64898/2026.09.21.753128v1.full",
  "id": "b0d61vp4ghxb0",
  "issns": [],
  "landing_url": "https://www.biorxiv.org/content/10.64898/2026.09.21.753128v1",
  "language": null,
  "licence": "CC-BY",
  "licence_label": "CC BY",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "biorxiv",
      "bioengineering"
    ],
    [
      "openalex",
      "1312|Chemical Synthesis and Analysis"
    ]
  ],
  "openalex": "W7213983510",
  "pdf_url": "https://www.biorxiv.org/content/10.64898/2026.09.21.753128v1.full.pdf",
  "pmcid": null,
  "pmid": null,
  "published": "2026-09-21",
  "publisher": null,
  "ris": "https://research.draconitic.com/paper/b0d61vp4ghxb0.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:23:40Z",
      "native_id": "10.64898/2026.09.21.753128",
      "source": "biorxiv",
      "url": "https://www.biorxiv.org/content/10.64898/2026.09.21.753128v1"
    }
  ],
  "subjects": [
    {
      "field": "biology",
      "origin": "biorxiv:bioengineering",
      "subfield": "systems-synthetic"
    }
  ],
  "title": "Random in vitro Protein single-Loop Engineering (RiPLE) by mRNA display",
  "title_text": "Random in vitro Protein single-Loop Engineering (RiPLE) by mRNA display",
  "type": "preprint",
  "updated_at": "2026-10-30T12:23:40Z",
  "url": "https://research.draconitic.com/paper/b0d61vp4ghxb0",
  "venue": "bioRxiv",
  "versions": []
}