Spotting-free Nano-Oscillator Array for Quantification of Virion Displayed Membrane Protein Binding Kinetics

ABSTRACT Transmembrane proteins, such as G-protein-coupled receptors (GPCRs), are critical for many cellular functions. They are also the most popular drug targets for various diseases, including cancer. For both understanding cellular functions and drug development, it is necessary to measure their binding activities with molecular ligands and drug candidates. However, this remains a difficult task because of several key challenges: 1) Directly measuring ligand interactions with native receptors on whole cells is greatly limited by low sensitivity and through put. 2) Methods to extract and purify transmembrane proteins often results in the loss of their native conformations, thereby resulting in dysfunction. 3) Extracted membrane proteins must be reconstituted in an artificial membrane environment that is tedious to synthesize and study. 4) Small molecules comprise ~90% of the current drugs, but their binding kinetics cannot be easily measured, often requiring indirect measurement approaches. 5) Current approaches lack sufficient throughput to service the needs of typically large drug libraries. This project aims to addresses these critical challenges with Spotting-Free Nano-Oscillator Array (SFNOA) technology, for high throughput, label-free quantification of ligand binding kinetics to transmembrane proteins in their native states. SFNOA achieves this feat by integrating several innovative technologies: 1) Virion displayed transmembrane proteins (VirD) preserve their native conformation and function while avoiding the drawbacks of extraction and reconstitution. 2) Nano-oscillator technology enables charge-sensitive plasmonic detection of label-free binding, making it particularly suitable for small molecule ligand measurements. 3) Affinity encoded multi-state DNA barcoding produces spotting-free sensor chips for multiplexed detection. 4) Plasmonic scattering microscopy enables high resolution detection of nano-oscillators for high density measurement. Each VirD is conjugated to a nano-oscillator, which oscillates under an alternating electric field. Upon binding of ligands to the transmembrane proteins, the oscillation amplitude changes and is measured precisely by a plasmonic imaging system, from which binding kinetics and affinity are quantified. Since the response signal scales with charge changes instead of mass changes, this direct detection method is particularly advantageous for small molecule ligands. Additionally, each VirD nano-oscillator is addressed with an affinity enhanced DNA barcode, making them rapidly readable in parallel for multiplexed and high throughput analysis. To showcase the significance and capabilities of SFNOA technology, small molecule drug interactions with various human GPCRs displayed on the viral envelopes of human herpes simplex virus-1 (HSV-1) will be measured. The goal of this fast-track STTR is to develop SFNOA for commercialization into a powerful high throughput platform for studying ligand binding kinetics to membrane proteins in their native states. SFNOA technology proposes to become a powerful new tool for high throughput study of membrane protein functions, minimizing missteps in early-stage drug discovery and screening, and quickly validating new therapies for rapidly evolving diseases.