GRIP Display: A One-Pot Library Display Platform for the Directed Evolution of Proteins

Library display technologies have enabled the development of peptides with affinity for a given substrate. Such affinity-capture reagents have driven progress in many fields, from basic biochemistry to neuropharmacology. A major limitation in the development of neuro-pharmaceuticals has been an inability to examine how the behavioral effects of drugs are mediated by each of the distinct yet intermingled cell types in any given brain region. DART (Drugs Acutely Restricted by Tethering) is the first method to overcome this technical barrier, enabling the delivery of therapeutics to a precise genetically defined neuronal cell type. At the core of DART’s specificity is a capture of a chemical Rx-HTL (HaloTag Ligand conjugated to a drug) by a genetically encoded HTP (HaloTag protein), creating an artificial dosing window. Although the technology has already revealed novel neurobiological insights, a narrow dosing window currently limits DART to neurobiological questions where dose can be tightly controlled, such as via intracranial infusion over a small brain volume. Our goal is to adapt the principles of directed evolution and library display to improve the dosing window of DART and enable its brain-wide delivery. Moreover, using the same principles, we aim to develop an orthogonal DART pair for multiplexed delivery of any combination of drugs to two distinct cell types. The underlying principle of a library display tool is a physical linkage between phenotype (a protein) and genotype (its corresponding nucleotide sequence). This conjugated mRNA, encoding the displayed protein, serves as a unique identifier for each variant. Over the past three decades, several display systems have been developed, each with a unique set of limitations. Typically, there is a tradeoff between the stability of this linkage and the number of unique variants (library size). Thus, no existing platform offers the desired trifecta of linkage stability, library size, and product yield. This work introduces a novel in vitro protein display technology called GRIP Display (Gluing RNA to Its Protein) that permits the generation and simultaneous screening of vast protein libraries (~10^14 variants) against a target of interest, with minimal genetic cross-talk, significant selection enrichment, and one-step simple experimental protocol. Here, we demonstrate 1) the development of GRIP Display and its utility in the optimization of a large binding tunnel of HTP to enhance the covalent capture of its chemical ligand; 2) the development of high-affinity orthogonal HTP/HTL pairs with minimal cross-reactivity; 3) a rational design of a novel peptide/RNA interaction to promote the avidity of binding and create a “single read” display technology GRIP.2. GRIP Display represents a valuable resource for the protein engineering community, and can substantially advance the range of neurobiological questions amenable to DART.