Aptamers that bind SARS-CoV-2 spike protein for COVID-19 rapid diagnostics and therapeutics

Abstract

The coronavirus disease 2019 (COVID-19) pandemic continues to severely impact healthcare and socioeconomic circumstances despite advances in rapid diagnostics and vaccines. One hurdle is the emergence of SARS-CoV-2 variants of concern, including the Omicron variant, which has enhanced infectivity and immune invasion. Improved rapid tests can limit viral spread by informing individuals of their infection status for personal risk assessment as well as disease surveillance. To that end, DNA aptamer-based rapid diagnostics show promise as cost-effective, shelf-stable alternatives to those based on antibodies. In the introduction, I discuss what DNA aptamers are, how they are discovered, and how they are applied to biosensing. In chapter 1, we selected for 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 x 105 copies/mL. In chapter 2, we describe a new aptamer that also binds wild-type spike protein at the NTD, named SARS-CoV-2 spike protein NTD-binding DNA aptamer 4 (SNAP4). SNAP4 binds with high affinity (< 30 nM) for SARS-CoV-2 spike protein, a 2-fold improvement over SNAP1. We utilized both SNAP1 and SNAP4 in an aptamer sandwich LFA (AptaFlow), which detected UV-inactivated intact SARS-CoV-2 virus at concentrations as low as 106 copies/mL. AptaFlow costs <$1 per test to produce, provides results in < 1 hour, and detects SARS-CoV-2 at concentrations that indicate higher viral loads and high probability of contagious transmission. In chapter 3, I will describe another new aptamer, SARS-CoV-2 Omicron RBD-binding aptamer (SCORe) which binds Omicron BA.1 and BA.2 RBD with nanomolar KD1. We employed aptamers SCORe.50 and SNAP4.74 in a multiplexed lateral flow assay (LFA) to distinguish between Omicron and wild-type spike protein at concentrations as low as 100 pM. Finally, we show that SCORe.50 and its dimerized form SCOReD can neutralize Omicron spike-pseudotyped virus infection of ACE2-overexpressing cells by >70%. Taken together, the aptamers SNAP1, SNAP4, and SCORe demonstrate great potential in low-cost, convenient, and rapid COVID-19 diagnostics.