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

Suttinee Poolsup; Emil Zaripov; Nico Hüttmann; Zoran Minic; Polina V Artyushenko; Irina A Shchugoreva; Felix N Tomilin; Anna S Kichkailo; Maxim V Berezovski

doi:10.1016/j.omtn.2023.02.010

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.

Authors

Suttinee Poolsup¹, Emil Zaripov¹, Nico Hüttmann², Zoran Minic², Polina V Artyushenko^{3

4

5}, Irina A Shchugoreva^{3

4

5}, Felix N Tomilin^{5

6}, Anna S Kichkailo^{3

4}, Maxim V Berezovski^{1

2}

Affiliations

¹ Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, ON K1N 6N5, Canada.
² John L. Holmes Mass Spectrometry Facility, Faculty of Science, University of Ottawa, Ottawa, ON K1N 6N5, Canada.
³ Laboratory for Digital Controlled Drugs and Theranostics, Federal Research Center "Krasnoyarsk Science Center SB RAS", Krasnoyarsk 660036, Russia.
⁴ Prof. V.F. Voino-Yasenetsky Krasnoyarsk State Medical University, Krasnoyarsk 660022, Russia.
⁵ Department of Chemistry, Siberian Federal University, Krasnoyarsk 660041, Russia.
⁶ Laboratory of Physics of Magnetic Phenomena, Kirensky Institute of Physics, Krasnoyarsk 660036, Russia.

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 (K_D 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.