DNA‐Directed Patterning for Versatile Validation and Characterization of a Lipid‐Based Nanoparticle Model of SARS‐CoV‐2

Abstract

Lipid‐based nanoparticles have been applied extensively in drug delivery and vaccine strategies and are finding diverse applications in the coronavirus disease 2019 (COVID‐19) pandemic—from vaccine‐component encapsulation to modeling the virus, itself. High‐throughput, highly flexible methods for characterization are of great benefit to the development of liposomes featuring surface proteins. DNA‐directed patterning is one such method that offers versatility in immobilizing and segregating lipid‐based nanoparticles for subsequent analysis. Here, oligonucleotides are selectively conjugated onto a glass substrate and then hybridized to complementary oligonucleotides tagged to liposomes, patterning them with great control and precision. The power of DNA‐directed patterning is demonstrated by characterizing a novel recapitulative lipid‐based nanoparticle model of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2)—S–liposomes—that presents the SARS‐CoV‐2 spike (S) protein on its surface. Patterning a mixture of S–liposomes and liposomes that display the tetraspanin CD63 to discrete regions of a substrate shows that angiotensin‐converting enzyme 2 (ACE2) specifically binds to S–liposomes. Subsequent introduction of S–liposomes to ACE2‐expressing cells tests the biological function of S–liposomes and shows agreement with DNA‐directed patterning‐based assays. Finally, multiplexed patterning of S–liposomes verifies the performance of commercially available neutralizing antibodies against the two S variants. Overall, DNA‐directed patterning enables a wide variety of custom assays for the characterization of any lipid‐based nanoparticle.

Detecting Intact Virus Using Exogenous Oligonucleotide Labels

“…In contrast to aCD63 beads, for which we observed no significant correlation ( r 2 = 0.81, p = 0.09), the fluorescence intensity of ACE2 beads was strongly correlated with a spike liposome number ( r 2 > 0.99, p = 0.0024). These data strongly support that cholesterol-oligonucleotides self-embed in the membrane of lipid bilayer nanoparticles, consistent with previous reports of cholesterol-based exosome and liposome labeling, , and that ACE2 beads can readily sequester lipid bilayer nanoparticles displaying spike protein for subsequent qPCR-based detection. The bead-based isolation of the nanoparticles is crucial to DIVER’s robustness to sample dilution.…”

“…As SARS-CoV-2 is an enveloped virus, we first employed a liposome model to verify the labeling of lipid-bilayer nanoparticles with synthetic oligonucleotides and their specific capture onto beads via the spike–ACE2 interaction. ACE2 is the primary receptor on human cells to which SARS-CoV-2 binds with high affinity − and has been used in several other SARS-CoV-2 technologies for virus trapping and detection. − The liposome model we used displayed the S1 subunit of the SARS-CoV-2 spike protein, which contains the receptor binding domain (RBD), in an orientation that binds to ACE2 (ref , hereafter referred to as spike liposomes). Spike liposomes were comparable in diameter (125.9 ± 27.3 nm; Figure S2a) to SARS-CoV-2.…”

“…DIVER is based on the following steps: (1) labeling enveloped viral particles with exogenous single-stranded DNA oligonucleotides; (2) capturing the labeled viral particles with paramagnetic beads coated with a virus-specific capture agent; and (3) performing qPCR on the oligonucleotide labels, which produces a signal that increases linearly with viral particle count (Figure a, Table S1). The oligonucleotide-labeling method in DIVER is adapted from one we previously applied to engineer cell–cell, cell–ligand, and liposome–protein interactions and to quantify tumor-derived extracellular vesicles (EVs) in biofluids…”

“…We developed and optimized DIVER using liposomes displaying the SARS-CoV-2 spike protein and validated our method using live spike-pseudotyped lentivirus. With these models, we show that DIVER is uniquely suited for sample pooling as it is more sensitive to the absolute number , rather than the concentration , of viral particles.…”

Bottom-up assembly of viral replication cycles

“…In fact, earlier studies have demonstrated how viruses, or parts thereof, can be assembled bottom-up and this ambitious approach has been recently transferred into the realm of SARS-CoV-2 research 21 , 22 . Intriguingly, in vitro reconstitution of the SARS-CoV-2 viral envelope and integration of the spike glycoprotein on lipid vehicles revealed how inflammatory fatty acids are exploited to trigger a molecular switch that couples local inflammation states to SARS-CoV-2 infectivity 23 – 25 .…”