Discovery and Characterization of Spike N-Terminal Domain-Binding Aptamers for Rapid SARS-CoV-2 Detection

Abstract

The coronavirus disease 2019 (COVID-19) pandemic has devastated families and disrupted healthcare, economies and societies across the globe. Molecular recognition agents that are specific for distinct viral proteins are critical components for rapid diagnostics and targeted therapeutics. In this work, we demonstrate the selection of novel DNA aptamers that bind to the SARS-CoV-2 spike glycoprotein with high specificity and affinity (<80 nM). Through binding assays and high resolution cryo-EM, we demonstrate that SNAP1 (SARS-CoV-2 spike protein N-terminal domain-binding aptamer 1) binds to the S N-terminal domain. We applied SNAP1 in lateral flow assays (LFAs) and ELISAs to detect UV-inactivated SARS-CoV-2 at concentrations as low as 5×10⁵ copies mL^-1 . SNAP1 is therefore a promising molecular tool for SARS-CoV-2 diagnostics.

Keywords: SARS-CoV-2; aptamers; coronavirus; cryo-EM; immunoassays.

Figure 1

Schematic of protein‐SELEX strategy using a DNA aptamer library. After an initial round of positive selection, subsequent rounds consisted of negative and positive selection with increasingly stringent conditions (Table S1). Negative selection included control proteins (BSA, MERS‐CoV S1, or SARS‐CoV S1) and protein immobilization platforms.

Figure 2

SNAP1 and SNAP3 aptamers bind to the NTD of SARS‐CoV‐2 S protein. A) Kinetic and binding equilibrium constants were measured by BLI. Biotinylated aptamer loaded on streptavidin biosensors was associated with SARS‐CoV‐2 S1 or NTD. Dotted lines indicate switch from analyte association to dissociation. K _D values (mean±s.d., n=5–6) were determined from a global fit of the kinetic data for a 1:1 binding model. B) Two views of the S/SNAP1/S2M11 complex cryo‐EM unsharpened map. SNAP1: purple, NTD: light blue, RBD: dark blue, rest of SARS‐CoV‐2 S: cyan, S2M11 CH (Fab fragment heavy chain): dark gray, S2M11 CL (Fab fragment light chain): light gray.

Figure 3

Aptamer‐based detection of UV‐inactivated SARS‐CoV‐2 virus. A) Schematic of HybriDetect LFA. B) Schematic of aptamer‐antibody sandwich ELISA. C,D) c mL⁻¹=copies per mL of virus. C) HybriDetect LFA strips were dipped in solutions of S protein (S), control lentivirus (LV), or UV‐inactivated SARS‐CoV‐2 virus (SC2) incubated with SNAP1.50. Top: representative image of developed strips. Bottom: quantification of band intensity. Bar graph shows mean and standard deviation of three replicates. Statistical comparison of LV, SC2, and S to none was determined by one‐way ANOVA with Bonferroni correction, *** denotes p<0.001, ** denotes p<0.01, and n.s. denotes no significance. D) ELISA using NS‐biotin or SNAP1‐biotin as capture agents to detect UV‐inactivated SARS‐CoV‐2 using anti‐S HRP antibody for detection. Bar graph shows mean and standard deviation of three replicates. Statistical comparison of NS to SNAP1 was determined by two‐way ANOVA with Fisher's LSD test, **** denotes p<0.0001, * denotes p<0.05, and n.s. denotes no significance.