Design and validation of a ribosome display library for synthetic nanobody selection

Single-domain antibodies (nanobodies) are compact, highly engineerable binding scaffolds widely used in structural biology, biotechnology, and therapeutics. When combined with the Legobody toolkit, they enable high-resolution cryo-electron microscopy (cryo-EM) analysis of small membrane proteins. Ribosome display is a powerful method for generating synthetic nanobodies (sybodies) with exceptionally high library diversity. However, existing sybody libraries are incompatible with the Legobody system, necessitating additional subcloning steps that reduce throughput and limit efficiency. Here, we report the rational design of a new ribosome-display-compatible nanobody library, termed S1.0, engineered for intrinsic compatibility with the Legobody platform through modifications in the C-terminal region. Compared to a benchmark sybody library, S1.0 features increased randomization in the complementarity-determining regions CDR1 and CDR3, while exhibiting reduced variability in CDR2. Selection against calmodulin demonstrated comparable efficiency to the benchmark library. Moreover, selection against thermostable green fluorescent protein (TGP) yielded multiple high-affinity sybodies, with nanomolar dissociation constants validated by size-exclusion chromatography and biolayer interferometry. Importantly, the selected sybodies are directly compatible with the Legobody system without requiring further engineering or subcloning. The S1.0 library represents a valuable resource for nanobody discovery and, in particular, provides an efficient route for generating Legobody-compatible binders suitable for structural studies of challenging small proteins by cryo-EM.