Single domain antibodies called nanobodies are excellent affinity reagents for membrane proteins. However, their generation relies on immunizations, which is only amenable to robust proteins and impedes selections in the presence of non-covalent or toxic ligands. To overcome these key limitations, we developed a novel in vitro selection platform, which builds on synthetic nanobodies called sybodies. Inspired by the shape diversity of natural nanobodies, three sybody libraries exhibiting different randomized surface shapes were engineered for high thermal stability. Using ribosome display, exceptionally large libraries were pre-enriched against membrane protein targets and subsequently funneled into a robust phage display process, thereby reducing selection bias. We successfully generated conformation-selective, high affinity sybodies against the human glycine transporter GlyT1, the human equilibrative nucleotide transporter ENT1 and a bacterial ABC transporter. Our platform builds exclusively on commercially available reagents and enables non-specialized labs to generate conformation-specific binders against previously intractable protein targets.

Synthetic single domain antibodies for the conformational trapping of membrane proteins