Three on Three: Universal and High-Affinity Molecular Recognition of the Symmetric Homotrimeric Spike Protein of SARS-CoV-2 with a Symmetric Homotrimeric Aptamer

Abstract

Our previously discovered monomeric aptamer for SARS-CoV-2 (MSA52) possesses a universal affinity for COVID-19 spike protein variants but is ultimately limited by its ability to bind only one subunit of the spike protein. The symmetrical shape of the homotrimeric SARS-CoV-2 spike protein presents the opportunity to create a matching homotrimeric molecular recognition element that is perfectly complementary to its structural scaffold, causing enhanced binding affinity. Here, we describe a branched homotrimeric aptamer with three-fold rotational symmetry, named TMSA52, that not only possesses excellent binding affinity but is also capable of binding several SARS-CoV-2 spike protein variants with picomolar affinity, as well as pseudotyped lentiviruses expressing SARS-CoV-2 spike protein variants with femtomolar affinity. Using Pd−Ir nanocubes as nanozymes in an enzyme-linked aptamer binding assay (ELABA), TMSA52 was capable of sensitively detecting diverse pseudotyped lentiviruses in pooled human saliva with a limit of detection as low as 6.3 × 10 3 copies/mL. The ELABA was also used to test 50 SARS-CoV-2-positive and 60 SARS-CoV-2-negative patient saliva samples, providing sensitivity and specificity values of 84.0 and 98.3%, respectively, thus highlighting the potential of TMSA52 for the development of future rapid tests.

Programmable Nanostructures Based on Framework-DNA for Applications in Biosensing

“…To overcome the limitation of the ability of monomeric aptamers to bind only one subunit of the spike protein. As shown in Figure 3 d, a triple rotationally symmetrical branching homo-trimeric aptamer has been developed for the symmetrical shape of the SARS-CoV-2 stinger protein [ 95 ]. This perfectly complementary structural scaffold greatly enhanced binding affinity.…”

“…; Yao, S.; et al Net-Shaped DNA Nanostructures Designed for Rapid/Sensitive Detection and Potential Inhibition of the SARS-CoV-2 Virus, 2022 , Copyright (2022), with permission from the American Chemical Society). ( d ) Complementarity of SARS-CoV-2 trimer spike proteins with trimer aptamers for molecular recognition [ 95 ]. (Reprinted from the Journal of the American Chemical Society, Li, J.; Zhang, Z.; Gu, J.; Amini, R.; Mansfield, A.G.; Xia, J.; White, D.; Stacey, H.D.…”

Recent development of nanotechnology-empowered antigen assay methods for the control of infectious diseases

“…The immunoassay established using this structure can detect pseudoviruses in human saliva samples with detection limits as low as 6.3 × 10 3 copies per mL. 18…”

“…54 These inorganic nanomaterials may be more catalytically active, more stable, and/or more convenient for synthesis and storage than natural enzymes, thus may help obtain enhanced detection sensitivity and/or lower cost. 18…”

“…2c). 9,[18][19][20][21][22] Thereby, the capture ability or selectivity for antigens can be further improved, ultimately achieving a PCR-like sensitivity. However, the dual antibody sandwich assay mode like in ELISA also requires multistep reagent addition and separation, which limits its convenience.…”