Camelid single-domain antibody fragments (“nanobodies”) provide the remarkable specificity of antibodies within a single 15 kDa immunoglobulin VHH domain. This unique feature has enabled applications ranging from use as biochemical tools to therapeutic agents. Nanobodies have emerged as especially useful tools in protein structural biology, facilitating studies of conformationally dynamic proteins such as G protein-coupled receptors (GPCRs). Nearly all nanobodies available to date have been obtained by animal immunization, a bottleneck restricting many applications of this technology. To solve this problem, we report a fully in vitro platform for nanobody discovery based on yeast surface display. We provide a blueprint for identifying nanobodies, demonstrate the utility of the library by crystallizing a nanobody with its antigen, and most importantly, we utilize the platform to discover conformationally-selective nanobodies to two distinct human GPCRs. To facilitate broad deployment of this platform, the library and associated protocols are freely available for non-profit research.

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Histone variant H2A.Z is a conserved feature of nucleosomes flanking protein-coding genes. Deposition of H2A.Z requires ATP-dependent replacement of nucleosomal H2A by a chromatin remodeler related to the multi-subunit enzyme, yeast SWR1C. How these enzymes use ATP to promote this nucleosome editing reaction remains unclear. Here we use single-molecule and ensemble methodologies to identify three ATP-dependent phases in the H2A.Z deposition reaction. Real-time analysis of single nucleosome remodeling events reveals an initial priming step that occurs after ATP addition that involves a combination of both transient DNA unwrapping from the nucleosome and histone octamer deformations. Priming is followed by rapid loss of histone H2A, which is subsequently released from the H2A.Z nucleosomal product. Surprisingly, rates of both priming and the release of the H2A/H2B dimer are sensitive to ATP concentration. This complex reaction pathway provides multiple opportunities to regulate timely and accurate deposition of H2A.Z at key genomic locations.

The LIN28:pre-let-7:TUTase ternary complex regulates pluripotency and oncogenesis by controlling processing of the let-7 family of microRNAs. The complex oligouridylates the 3′ ends of pre-let-7 molecules, leading to their degradation via the DIS3L2 exonuclease. Previous studies suggest that components of this complex are potential therapeutic targets in malignancies that aberrantly express LIN28. In this study we developed a functional epitope selection approach to identify nanobody inhibitors of the LIN28:pre-let-7:TUT4 complex. We demonstrate that one of the identified nanobodies, Nb-S2A4, targets the 106-residue LIN28:let-7 interaction (LLI) fragment of TUT4. Nb-S2A4 can effectively inhibit oligouridylation and monouridylation of pre-let-7g in vitro. Expressing Nb-S2A4 allows maturation of the let-7 species in cells expressing LIN28, highlighting the therapeutic potential of targeting the LLI fragment.