Integrating seed-based design with ribosome display for the development of nanobody-like protein scaffolds

Protein scaffolds are indispensable in biology and bioengineering, providing stable, engineerable frameworks for diverse molecular applications. However, existing methods are limited in structural diversity and require extensive experimental or computational resources. Here, we present an integrated strategy that combines seed-based framework design with ribosome display to generate novel nanobody-like protein scaffolds. Sequence alignment of nanobody frameworks defined four conserved 10-residue seed frameworks, which were embedded in random sequences to generate a synthetic library exceeding 10¹³ variants. Iterative ribosome display selection coupled with next-generation sequencing, and subsequent structural prediction using AlphaFold 3 indicated that top candidates adopt canonical nanobody-like folds. Two candidates were stably expressed and exhibited good thermal stability, with Tm around 63.2 °C, validating the functional feasibility of the scaffolds generated by this pipeline. This work establishes a proof-of-concept scaffold-generation paradigm that bypasses reliance on natural templates or purely computational design, offering a streamlined and efficient route.