ABSTRACT
The state-of-the-art SARS-CoV-2 detection methods include qRT-PCR and antibody-based lateral flow assay (LFA) point-of-care tests. Despite the high sensitivity and selectivity, qRT-PCR is slow, expensive and needs well-trained operators. On the other extreme, LFA suffers from low sensitivity albeit its fast detection speed, low detection cost and ease of use. Therefore, the continuing COVID-19 pandemic calls for a SARS-CoV-2 detection method that is rapid, convenient and cost-effective without compromise in sensitivity. Here we provide a proof-of-principle demonstration of an optimized aptamer-based nanointerferometer that enables rapid and amplification-free detection of SARS-CoV-2 spike protein-coated pseudovirus directly from human saliva with the limit of detection (LOD) of about 400 copies per mL. This LOD is on par with that of qRT-PCR, making it 1000 to 100,000-fold more sensitive than commercial LFA tests. Using various combinations of negative selections during the screens for the aptamer targeting the receptor binding domain of the spike protein of SARS-CoV-2, we isolated two aptamers that can distinguish the Omicron and Delta variants. Integrating these two aptamers with LFA strips or the nanointerferometer sensors allows both detection and differentiation of the Omicron and Delta variants which has the potential to realize rapid triage of patients infected different SARS-CoV-2 variants.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , COVID-19/diagnosis , Pandemics , OligonucleotidesABSTRACT
The spread of COVID-19 has affected billions of people across the globe, and the diagnosis of viral infection still needs improvement. Because of high immunogenicity and abundant expression during viral infection, SARS-CoV-2 nucleocapsid (N) protein could be an important diagnostic marker. This study aimed to develop a label-free optical aptasensor fabricated with a novel single-stranded DNA aptamer to detect the N protein. The N-binding aptamers selected using asymmetric-emulsion PCR-SELEX and their binding affinity and cross-reactivity were characterized by biolayer interferometry. The tNSP3 aptamer (44 nt) was identified to bind the N protein of wild type and Delta and Omicron variants with high affinity (KD in the range of 0.6-3.5 nM). Utilizing tNSP3 to detect the N protein spiked in human saliva evinced the potential of this aptamer with a limit of detection of 4.5 nM. Mass spectrometry analysis was performed along with molecular dynamics simulation to obtain an insight into how tNSP3 binds to the N protein. The identified epitope peptides are localized within the RNA-binding domain and C terminus of the N protein. Hence, we confirmed the performance of this aptamer as an analytical tool for COVID-19 diagnosis.
ABSTRACT
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created an urgent need for new technologies to treat COVID-19. Here we report a 2'-fluoro protected RNA aptamer that binds with high affinity to the receptor binding domain (RBD) of SARS-CoV-2 spike protein, thereby preventing its interaction with the host receptor ACE2. A trimerized version of the RNA aptamer matching the three RBDs in each spike complex enhances binding affinity down to the low picomolar range. Binding mode and specificity for the aptamer-spike interaction is supported by biolayer interferometry, single-molecule fluorescence microscopy, and flow-induced dispersion analysis in vitro. Cell culture experiments using virus-like particles and live SARS-CoV-2 show that the aptamer and, to a larger extent, the trimeric aptamer can efficiently block viral infection at low concentration. Finally, the aptamer maintains its high binding affinity to spike from other circulating SARS-CoV-2 strains, suggesting that it could find widespread use for the detection and treatment of SARS-CoV-2 and emerging variants.