Discovery of DNA aptamers targeting SARS-CoV-2 nucleocapsid protein and protein-binding epitopes for label-free COVID-19 diagnostics

Mol Ther Nucleic Acids. 2023 Mar 14:31:731-743. doi: 10.1016/j.omtn.2023.02.010. Epub 2023 Feb 14.

Abstract

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.

Keywords: COVID-19 diagnosis; MT: Oligonucleotides: Diagnostics and Biosensors; SARS-CoV-2 nucleocapsid detection; aptamer selection; binding motif identification; biolayer interferometry; label-free optical aptasensor.