Nucleocapsid protein binding DNA aptamers for detection of SARS-COV-2

Abstract

The severe acute respiratory syndrome coronavirus (SARS-CoV-2) has infected millions of individuals and continues to be a major health concern worldwide. While reverse transcription-polymerase chain reaction remains a reliable method for detecting infections, limitations of this technology, particularly cost and the requirement of a dedicated laboratory, prevent rapid viral monitoring. Antigen tests filled this need to some extent but with limitations including sensitivity and specificity, particularly against emerging variants of concern. Here, we developed aptamers against the SARS-CoV-2 Nucleocapsid protein to complement or replace antibodies in antigen detection assays. As detection reagents in ELISA-like assays, our DNA aptamers were able to detect as low as 150 pg/mL of the protein and under 150 k copies of inactivated SARS-CoV-2 Wuhan Alpha strain in viral transport medium with little cross-reactivity to other human coronaviruses (HCoVs). Further, our aptamers were reselected against the SARS-CoV-2 Omicron variant of concern, and we found two sequences that had a more than two-fold increase in signal compared to our original aptamers when used as detection reagents against protein from the Omicron strain. These findings illustrate the use of aptamers as promising alternative detection reagents that may translate for use in current tests and our findings validate the method for the reselection of aptamers against emerging viral strains.

Keywords: Antigen; Aptamer; COVID-19; Diagnostic; Nucleocapsid; SARS-CoV-2.

Graphical abstract

Fig. 1

Selection of SARS-CoV-2 Nucleocapsid Protein Aptamers using Melting-Off Based Library Enrichment. A. Library recovery following a melting-off based library enrichment determined as a ratio between material recovered from the selection or parallel assessment step and the input amount of material. Specific selection conditions are indicated below the bar graph. B. Bioinformatics based analysis predicted secondary structures (top panels) and sequence data (bottom panels) for the five aptamer candidates. The secondary structure and free energy for each aptamer was computed by Quikfold 2.3 (Zuker 2003) at 23 °C, 150 mM Na+ (combined monovalent cations in 1X SELEX Buffer). Sequences displayed below each structure. PCR primer annealing regions (5′- CGA GGC TCT CGG GAC GAC -[sequence]- GTC GTC CCG CCT TTA GGA TTT ACA G −3′) are highlighted in bold. C. Aptamers identified through SELEX were evaluated for their binding to recombinant nucleocapsid protein from HCoVs (200 ng/mL) using a direct ELASA format and compared to a commercially available monoclonal antibody. Background was subtracted and error bars indicate standard deviation. Statistical significance was reached for all conditions of Alpha variant compared to other HCoV.

Fig. 2

Aptamers as detection reagents for SARS-CoV-2 N protein and inactivated virus in a hybrid ELASA format. Aptamers 1 and 5 were evaluated as detection reagents in a hybrid ELASA format where an antibody was used for capture and aptamers were used as detection reagents. A. Detection of SARS-CoV-2 Alpha recombinant nucleocapsid protein or B. inactivated virus. C. Aptamers were evaluated for their ability to detect N protein from SARS-CoV-2 viral strains of interest (200 ng). D. Detection of SARS-CoV-2 Omicron recombinant nucleocapsid protein or E. inactivated virus. The error bars indicate standard deviation and statistical significance is indicated when p < 0.05 and was calculated using one-way ANOVA with Šidák multiple comparisons test (Figures A, B, D and E) and Welches T Test (Figure C).

Fig. 3

Identification of aptamers reselected against N protein from Omicron SARS-CoV-2 variant. The top 200 aptamers selected against the alpha variant were reselected against the omicron variant using a microarray assay. A. Top 2 Aptamer candidates based on highest binding affinity against SARS-CoV-2 Omicron Nucleocapsid Protein. B. Aptamers were evaluated for their binding to N protein from SARS-CoV-2 viral strains of interest (200 ng) using a hybrid ELASA format. C. Reselected aptamers were evaluated for their binding to recombinant nucleocapsid protein from HCoVs (200 ng/mL) using a direct ELASA format. Background was subtracted and error bars indicate standard deviation. D. Relative binding of aptamers as detection reagents for SARS-CoV-2 Omicron recombinant nucleocapsid protein. The error bars indicate standard deviation and statistical significance is indicated when p < 0.05 and was calculated using one-way ANOVA with Šidák multiple comparisons test (Figure D) with and two-sided t-tests with Welch’s correction (Figure B).

Fig. 4

Screening of aptamer pairs for the detection of SARS-CoV-2 N protein. Thiolated capture aptamer was analyzed in combination with each of the biotinylated detection aptamers using 400 to 50 ng/mL of SARS-CoV-2 N recombinant protein. The graph title refers to the biotinylated aptamer used as detection (or detection monoclonal antibody), and the colors indicate the thiolated capture aptamer (or capture antibody). The error bars indicate standard deviation and statistical significance is indicated when p < 0.05 and was calculated using one-way ANOVA.