High-Throughput Functional Screening with DNA Nanoswitches

Our understanding of underlying disease biology and the investigation and identification of potential drug targets has been accelerated with advances in sequencing, bioinformatics and the “omics” paradigm alongside the generation of new and sensitive research tools such as RNAi, CRISPR, and many others. In parallel, the ability to capture high resolution structures of molecules, and generate large chemical and biologic libraries to probe potential drug candidates has grown considerably. However, current approaches for therapeutic screening often limited in either molecular diversity, or the ability to assay complex modes of drug-target interaction. High-throughput screening of large chemical libraries can interrogate varied molecular or phenotypic outputs from FRET to genetic reporters. However, libraries cannot be combined without information loss, they must be massively parallelized using plate-based methods, limiting the number of compounds that can be screened. Nucleotide-encoded chemical or biologic libraries (such as those used in phage, yeast, ribosome, and mRNA display) can generate immense libraries of multiple different potential binder types that can mimic the diversity found in nature. Still, screening of these libraries is often limited to simple binding assays, which can make it difficult to select for compounds based on their functional properties. Thus, with current approaches in drug discovery, it can be challenging to find binders against targets with non-enzymatic or complex functions, or with allosteric or enhancing capabilities. To overcome some of these limitations and create a “best of both worlds” approach iv screening assay, we have developed Nanoswitch-display, a method incorporating both a nucleotide encoded nanobody library by mRNA-display, and DNA Nanoswitches, a simple DNA device that can measure multiplexes of biomolecules that are functionally consequential. Using the SARS-CoV-2 spike protein and its receptor, ACE2, we first validated our methodology and constructed a nanobody mRNA-display library compatible with DNA nanoswitches. We generated and validated high affinity single target binders against the SARS-CoV-2 Beta variant receptor binding domain of the spike protein. We next used Nanoswitch-display to screen for nanobodies that directly or allosterically enhanced the spike:ACE2 interaction. Nb1, discovered from our screen, dramatically increased both the biochemical affinity of the interaction, and infectivity of spike-pseudovirus in cells. This work shows the potential of a nanoswitch display assay to generate multi-targeting or functionally specific nanobodies as potential future therapeutics and opens the