Structure-Guided Development of Bivalent Aptamers Blocking SARS-CoV-2 Infection

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused devastation to human society through its high virulence, infectivity, and genomic mutations, which reduced the efficacy of vaccines. Here, we report the development of aptamers that effectively interfere with SARS-CoV-2 infection by targeting its spike protein, which plays a pivotal role in host cell entry of the virus through interaction with the viral receptor angiotensin-converting enzyme 2 (ACE2). To develop highly effective aptamers and to understand their mechanism in inhibiting viral infection, we determined the three-dimensional (3D) structures of aptamer/receptor-binding domain (RBD) complexes using cryogenic electron microscopy (cryo-EM). Moreover, we developed bivalent aptamers targeting two distinct regions of the RBD in the spike protein that directly interact with ACE2. One aptamer interferes with the binding of ACE2 by blocking the ACE2-binding site in RBD, and the other aptamer allosterically inhibits ACE2 by binding to a distinct face of RBD. Using the 3D structures of aptamer–RBD complexes, we minimized and optimized these aptamers. By combining the optimized aptamers, we developed a bivalent aptamer that showed a stronger inhibitory effect on virus infection than the component aptamers. This study confirms that the structure-based aptamer-design approach has a high potential in developing antiviral drugs against SARS-CoV-2 and other viruses.

Sensing Characteristics of SARS-CoV-2 Spike Protein Using Aptamer-Functionalized Si-Based Electrolyte-Gated Field-Effect Transistor (EGT)

“…The aptamer was developed by VIRO-SELEX [ 25 , 26 ]. Several cycles of VIRO-SELEX were performed, and a pool of 11 cycles was chosen to analyze the sequence [ 27 ].…”

Structural Insights into Protein–Aptamer Recognitions Emerged from Experimental and Computational Studies

“…Monomeric proteins may be simultaneously targeted by two aptamers, thus forming ternary complexes (1ˆ:2 or 1:2 in Table 1). In particular, eight structures of ternary complexes involving either thrombin (entries #62, 63, 104, and 129-131 PDB IDs 5EW1, 5EW2, 7NTU, 7ZKM, 7ZKN, and 7ZKO) [82,107,114] or the spike protein S1 from SARS-CoV-2 (entries #143 and 144, PDB IDs 8J1Q and 8J26) [119] have been reported until now.…”

“…Interestingly, the analysis of the interacting surface of TBA and thrombin at exosite II, which is non-canonical for this aptamer, indicated that it is similar to the interface area of TBA at exosite I and different from the surfaces commonly detected in the complexes with HD22_27mer and Toggle-25t aptamers targeting exosite II with high affinity (Figure S3). Finally, in the last year, the cryo-EM structures of two 1:2 ternary complexes (entries #143 and 144, PDB IDs 8J1Q and 8J26) [119] in which the receptor-binding domain (RBD) of SARS-CoV-2 spike protein S1 is simultaneously bound to two different aptamers, AM032-0 and AM047-0, or their derivatives, at two distinct binding sites, were reported (Figure 6c). These studies showed that the binding of angiotensin-converting enzyme 2 (ACE2), the viral receptor protein of the host cell, to the spike protein S1 is hindered by the AM032 aptamer family.…”

“…It has to be noted that in these structures, the protein is also bound to the Fab domain of the imdevimab antibody used to increase the overall size of the complexes thus making them suitable for cryo-EM studies. These cryo-EM structures represented the starting models to design a bivalent aptamer that would strongly inhibit SARS-CoV-2 pseudovirus infection [119].…”