Engineering functionally-optimized aptamers against SARS-Cov-2 for blocking spike-ACE2 interaction and aptasensor detection

Abstract

Both the limited research about structure-function relationship and the ill-defined process of conformational dynamic change greatly impede the development of aptamer engineering transformation and seriously restrict the practical applications of aptamers. In this work, an optimization strategy combining exonuclease III (Exo III) digestion and in silico simulation was presented for the first time for constructing high-affinity and functional aptamers and clarifying the three-dimensional (3D) structure of aptamer-target complexes and the conformational dynamic conversion in the process of aptamer recognizing its target. As a demonstration, the parent aptamer (Apt2) against SARS-CoV-2 spike subunit 1 (S1) was mutated or truncated at the predicted binding sites to produce eight derivatives (Seq1-Seq8). The progeny Seq3 exhibited a higher affinity for S1 and a better blocking effect on S1-angiotensin-converting enzyme 2 (ACE2) interaction compared to Apt2. Subsequently, Seq3 sealed the pores of nickel-doped zeolitic imidazolate framework-8 (NZIF-8) loaded with Rhodanine (Rho) to fabricate the aptasensor (NZIF-8-Rho-Apt) for inactivated virus detection, showing excellent performances in spiked actual samples. Therefore, this post systematic evolution of ligands by exponential enrichment (post-SELEX) is a very effective and general strategy for acquiring functionally-optimized aptamers.

Keywords: Aptamer optimization; Aptasensors; Exonuclease III digestion; In silico simulation; Inactivated virus detection; SARS-CoV-2 spike.

Graphical abstract

Fig. 1

The non-denatured PAGE of the digestion products and the prediction of interaction sites between Apt2 and RBD. (a) S1-inducible Exo III digestion of Apt2. The final concentration of Apt2, Exo III and S1 was 0.5 μM, 0.5 U/μL and 1 μM, respectively. (b) Semi-quantitative analysis of residual Apt2. The group that contained only Apt2 was normalized to 1. The bar chart was represented by mean ± standard deviation (M ± SD), and the relative standard deviation (RSD) = SD/M. The RSD of each reaction group from left to right was 1.26 %, 0.68 %, 2.75 %, 3.53 %, and 1.34 %, respectively. (c) The predicted secondary structure of Apt2 by Mfold web server (△G = −7.01 kcal/mol). (d) The natural PAGE characterization of the reaction products to investigate the S1-induced digestive effect of Exo III on Apt2 at different enzyme concentrations, and the final concentration of Apt2 and S1 remained constant at 0.5 μM and 1 μM, respectively. (e) Semi-quantitative analysis of residual Apt. The group without Exo III digestion was normalized to 1 (∗p < 0.05, and NS represented no significance), and the RSD of each reaction group from left to right was 0.51 %, 1.17 %, 0.52 %, 2.26 %, 1.44 % and 2.31 %, respectively. The 3D structure simulation of Apt2 with scores: 26.54 (f), the 3D structure of RBD domain (223 amino acid) from Protein Data Bank (g) and the molecular docking between Apt2 and RBD with the lowest docking energy score: 358.12 (h). The predicted four interacting binding sites (I, II, III and IV) was shown in (c).

Fig. 2

The natural PAGE of the digestive residual and the binding affinity of aptamer to S1 by ELONA. Seq1 (a) and Seq3 (b) were digested by Exo III in the absence or presence of S1 to produce of the shallow and short residue bands. Compared to the S1-induced digestive effects on Apt2, it was interesting that the S1-induced digestion sensitivity to aptamer was reversed in the Seq2 (c and e) and Seq4 (d and f). The bar chart was represented by mean ± standard deviation (M ± SD), and the relative standard deviation (RSD) = SD/M. The RSD of each reaction group in (e) and (f) from left to right was 0.02 %, 0.36 %, 0.86 %, 0.06 %, 0.19 % and 0.34 %, respectively. The final concentration of aptamer, Exo III and S1 remained 0.5 μM, 0.5 U/μL and 1 μM, respectively. (g) Schematic diagram of affinity detection by ELONA. (h) The Seq2 and Seq3 exhibited a higher affinity for S1 than Apt2. The Seq1 and Seq4 showed a lower affinity for S1 than Apt2.

Fig. 3

(a) Schematic diagram of the competitive binding efficiency of ACE2 to S1-binding aptamers by ELONA. (b) The competitive binding abilities of the Apt2, Seq2 and Seq3 to ACE2 were 82 %, 87 % and 77 %, respectively. The RSD of each group from left to right was 0.90 %, 0.85 %, 1.23 %, 0.41 %, 0.74 %, and 1.02 %, respectively. (c) Schematic diagram of ELONA detection of the aptamer-mediated blockade of RBD-ACE2 interaction. (d) Seq3 (X0, namely IC50 = 6.28 nM) presented the best neutralization efficiency compared to Apt2 (IC50 = 13.07 nM) and Seq2 (IC50 = 20803.39 nM).

Scheme 1

(a) Schematic illustration of engineering construction of the aptamer against SARS-CoV-2 with high affinity and blocking efficiency based on Exo III digestion and in silico post-SELEX. (b) Schematic diagram of establishing the ratiometric fluorescent aptasensor for the detection of InCov-2.

Fig. 4

The characterization of NZIF-8 nanomaterials. (a) TEM, (b) SEM, (c) PXRD spectra compared to the ZIF-8 simulation, (d) high-angle annular dark-field scanning element mapping images under TEM for observing the spatial distribution of C, N, O, Ni and Zn elements in the NZIF-8 and (e) EDS.

Fig. 5

(a) Fluorescence emission spectrum of Rho, NZIF-8-Rho, NZIF-8-Rho-Apt and NZIF-8. (b) UV–vis spectra of Rho and the supernatant from NZIF-8-Rho and NZIF-8-Rho-Apt. The hydrodynamic diameters and zeta-potential values of (c and e) NZIF-8 and (d and f) NZIF-8-Rho-Apt were evaluated by dynamic light scattering. (g) Fluorescence spectra of the NZIF-8-Rho-Apt aptasensor incubated with various concentrations of InCov-2. (h) Linear regression curves of the FEm-max/F459 ratio for the various InCov-2 concentrations.