The workflow of the CAAMO framework for designing high-affinity RNA aptamers and its application in the development of novel RNA aptamers targeting the RBD of the SARS-CoV-2 spike protein.

(A) Schematic diagram of the spike protein (trimer) of SARS-CoV-2 virus with one of the three RBD regions highlighted in gold. (B) The sequence and Mfold-predicted secondary structure of a SELEX-derived RNA aptamer termed Ta targeting the RBD of SARS-CoV-2 spike protein, which was the initial input information of the CAAMO framework. (C) Schematic diagrams showing that high-affinity RNA aptamers optimized through the CAAMO framework can complement existing antibody-neutralizing treatments for COVID-19. Antibodies (purple) and RNA aptamers (yellow) that bind to the RBDs of the SARS-CoV-2 spike protein can neutralize viral infection by blocking its interaction with the human ACE2. The designed aptamers (marked by red dots) with binding affinities comparable to antibodies can further strengthen the neutralizing treatment when antibody escape occurs. (D) Illustrative workflow of the CAAMO framework by integrating computational techniques with experimental validation. (E) A representative output after CAAMO framework optimization (the aptamer TaG34C) and its comparison with the original Ta sequence. RNA aptamer is colored by pink and RBD is shown in dark blue surface. The mutated nucleotides (G34 in wild type Ta and C34 in optimized TaG34C) are highlighted in pink sticks, atoms oxygen and nitrogen are colored by red and blue, respectively. (F) Competitive binding experiments to compare the binding capabilities of RNA aptamers Ta or TaG34C and a commercial neutralizing antibody (SinoBiological, Cat: 40592-R001) to the RBD of SARS-CoV-2 spike protein. Gradient amount of the commercial neutralizing antibody was titrated into the buffer containing 5 μM aptamer (Ta or TaG34C) and 40 μM RBD. The intensity of aptamer-RBD bands were quantified with Image J and normalized to that of the mixture without antibody, which was set to 100%. Data were collected from the images of EMSA shown in Fig. 5D.

Determination of the binding model of the aptamer Ta and the RBD of the SARS-CoV-2 spike protein via a multi-strategy approach.

(A) Flowchart illustrating the combination of RNA 3D structure prediction, ensemble docking and clustering, and binding capacity assessment by MM/GBSA and steered MD to determine the most probable binding conformation of the aptamer Ta to the RBD. (B) Six main binding modes, i.e., conformations 01-06, with clearly distinct binding conformations after ensemble docking and clustering for further binding ability assessment. The RBD and the aptamer Ta are shown in dark blue and dark red, respectively. For clarity, the surface of RBD is also displayed. For the aptamer Ta, conformations 01-06 are named in a descending order of their respective cluster sizes and their colors are lightened gradually. (C) The binding energies between the aptamer Ta and RBD estimated by MM/GBSA method for six binding candidates shown in plane (B). Data represent mean±s.d. collected from three independent calculations. (D) The rupture works required to separate the bound aptamer Ta from the RBD for different binding conformation candidates. Data were collected from four independent steered MD simulations. Limited by the available computing resources, only the first four binding conformations were assessed. The orange line is the median, boxes extended form lower to upper quartiles, whiskers showing the range of nonoutlier data. (E) Overview of the most probable binding model (conformation 01) of the aptamer Ta and RBD. The RBD is shown in cyan cartoon representation, the apical loop, bulge part, and end stem of the aptamer Ta are colored plum, pink, and sandy brown, respectively. The key residues of the RBD and nucleotides of the aptamer Ta for binding interactions are shown in sticks, all nitrogen atoms colored by blue, and all oxygen atoms by red. (F) EMSA results of the aptamer Ta (upper panel) and the weakly binding aptamer Tc (negative control, lower panel) bound to the RBD of the SARS-CoV-2 spike protein and (G) the resultant binding curve for the aptamer Ta. The dissociation constant (Kd) was calculated from the EMSA image quantification from three independent experiments.

The aptamer Ta exhibits a comparable binding capability to RBD compared to neutralizing antibodies.

(A) Contact ratios of residues on the RBD by ACE2 (derived from MD simulations), the aptamer Ta (derived from MD simulations), and neutralizing antibodies (derived from all available crystal structures deposited in PDB). For reference, the electrostatic potential distribution on the RBD surface generated by PyMOL (version 2.3.5) program was also shown. (B) Key residues on RBD (with contact ratio larger than 0.5 in plane (A)) contacted by ACE2, the aptamer Ta, and neutralizing antibodies were displayed as a Venn diagram. (C) Binding energies estimated by MM/GBSA calculations for ACE2, the aptamer Ta, and three representative antibodies (P2C-1F11, 2H2, and S2E12) binding to RBD. (D) Binding ability of a commercial antibody (40592-R001) to RBD was examined by a protein binding assay. Three micrograms of RBD protein and 3 μg of the antibody individually or mixed together were run a native-PAGE gel, which then was stained with Coomassie brilliant blue. (E) The RBD binding abilities of the aptamer Ta and commercial antibody 40592-R001 were compared by EMSA competitive binding experiments. The aptamer-RBD complex bands were shown after running on an agarose gel following the incubation of 40 μM RBD protein, 5 μM aptamer Ta and the antibody 40592-R001.

Structure-based rational design of Ta analogues with improved binding affinities and their experimental validation.

(A) Flowchart illustrating the combination of rational mutation scanning, secondary structure analysis (SSA), and free energy perturbation (FEP) to optimize aptamer binding affinities. (B) Contact ratios of nucleotides on the aptamer Ta bound to RBD (dark blue surface). Data were collected from three independent MD simulations. (C) SSA based on BP similarity for 16 selected nucleotides mutated to other three bases. Definition of BP similarity was given in the main text. Only mutations with BP similarity greater than 0.9 were subjected to further FEP calculations. (D) The binding free energy changes assessed by FEP calculations for selected single mutations. Data represent mean±s.d. collected from five independent simulations. (E) EMSA results of Ta, Tc, and 6 designed candidate sequences bound to the RBD of SARS-CoV-2 spike protein. The aptamer-RBD complex bands were detected by running an agarose gel after incubation of 40 μM of RBD protein with 5 μM indicated aptamer variant. (F) Comparison of the binding free energy changes derived from FEP calculations (ΔΔG, left scale) and EMSA experiments (ΔΔGexp, right scale) for 6 designed candidate aptamers. Data represent mean±s.d. collected from 5 (FEP)/three (EMSA) independent replications.

Designed aptamer TaG34C showed superior binding ability than WT Ta or antibody.

(A) Binding curves and the resultant dissociation constants (Kd) for WT Ta and the designed TaG34C with RBD proteins. The Kd values were calculated from the EMSA image quantification with s.d. from three independent experiments. (B) Comparison of the Ta-RBD and TaG34C-RBD binding complexes. RNA is shown as a dark red ribbon while RBD is displayed as a dark blue surface. Zoom-in is the nucleotide G34 (WT Ta)/C34 (TaG34C) and its surrounding nucleotide U35 and residue PHE486 shown in sticks. Atoms oxygen and nitrogen are colored in red and blue, respectively, and carbon atoms are colored by their locations. Definitions of two related distances (D1 and D2) are also displayed. (C) Violin plots of distributions of two related distances (D1 and D2) between selected RNA nucleotide-protein residue in Ta-RBD and TaG34C-RBD binding complexes, respectively. Data were collected from three independent MD simulations. (D) EMSA images of competitive binding experiments to characterize the RBD binding abilities of RNA aptamers (WT Ta and TaG34C) and the commercial monoclonal SARS-CoV-2 neutralizing antibody 40592-R001. The aptamer-RBD complex bands were showed by running an agarose gel after incubation of 40 μM of RBD protein and indicated aptamer with varying concentrations of the antibody 40592-R001. Results showed that TaG34C, but not WT Ta, exhibited a higher binding affinity to the RBD proteins than that of the antibody.