Random in vitro Protein single-Loop Engineering (RiPLE) by mRNA display
- 1The University of Tokyo
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.
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BibTeX
@misc{colas2026random,
title = {{Random in vitro Protein single-Loop Engineering (RiPLE) by mRNA display}},
author = {K. Colas and T. Ko and G. Philippe and H. Suga},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.21.753128},
url = {https://www.biorxiv.org/content/10.64898/2026.09.21.753128v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Random in vitro Protein single-Loop Engineering (RiPLE) by mRNA display
AU - K. Colas
AU - T. Ko
AU - G. Philippe
AU - H. Suga
PY - 2026
DA - 2026/09/21
PB - bioRxiv
DO - 10.64898/2026.09.21.753128
UR - https://www.biorxiv.org/content/10.64898/2026.09.21.753128v1
AB - 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.
ER -