Generation and characterization of nucleic acid aptamers targeting the capsid P domain of a human norovirus GII.4 strain

Abstract

Human noroviruses (NoV) are the leading cause of acute viral gastroenteritis worldwide. Significant antigenic diversity of NoV strains has limited the availability of broadly reactive ligands for design of detection assays. The purpose of this work was to produce and characterize single stranded (ss)DNA aptamers with binding specificity to human NoV using an easily produced NoV target-the P domain protein. Aptamer selection was done using SELEX (Systematic Evolution of Ligands by EXponential enrichment) directed against an Escherichia coli-expressed and purified epidemic NoV GII.4 strain P domain. Two of six unique aptamers (designated M1 and M6-2) were chosen for characterization. Inclusivity testing using an enzyme-linked aptamer sorbent assay (ELASA) against a panel of 14 virus-like particles (VLPs) showed these aptamers had broad reactivity and exhibited strong binding to GI.7, GII.2, two GII.4 strains, and GII.7 VLPs. Aptamer M6-2 exhibited at least low to moderate binding to all VLPs tested. Aptamers significantly (p<0.05) bound virus in partially purified GII.4 New Orleans outbreak stool specimens as demonstrated by ELASA and aptamer magnetic capture (AMC) followed by RT-qPCR. This is the first demonstration of human NoV P domain protein as a functional target for the selection of nucleic acid aptamers that specifically bind and broadly recognize diverse human NoV strains.

Human Norovirus Aptamer Exhibits High Degree of Target Conformation-Dependent Binding Similar to That of Receptors and Discriminates Particle Functionality

“…The docking simulation predictions were confirmed by mutational analysis of aptamer M6-2. Native M6-2 exhibited binding, considered a VLP/no-VLP ratio of >2.0 per convention (8, 19, 21), for all of the aptamer concentrations tested, except at 0.001 µM. None of the other mutated aptamers displayed binding at any concentration, confirming docking simulation predictions.…”

“…A previously performed motif analysis was also used to identify a general interacting region of the ssDNA aptamer (Fig. 4A) (19). An earlier study reported docking between an ssDNA aptamer and the structure of the entire norovirus VP1 protein (22).…”

“…The assays and analyses used are easily adaptable to other targets, as aptamers have been successfully used to detect infectious virus in stool samples (19, 21) with minimal modification. The most recent strain of the pandemic human norovirus genotype that causes the majority of human norovirus cases (GII.4 Sydney) was chosen as the model for this proof-of-concept study.…”

“…One monoclonal antibody, NS14, has shown promise as a broadly reactive reagent for the GII genogroup of noroviruses (16, 18) and is thus an improvement on HBGAs. Aptamer M6-2, generated against a GII.4 human norovirus strain, has been found to react to GI and GII human noroviruses, binding to all of the noroviruses tested in a panel of 14 human norovirus strains (19). We hypothesized that nucleic acid aptamers may be an alternative ligand for use in infectivity discrimination.…”

“…They offer the advantages of low cost; ease of synthesis, purification, and functional modification; and high stability. Multiple aptamers have been generated for human norovirus binding, some of which are broadly reactive (19–22). Evidence has been presented that aptamers tend to bind their targets with multiple different discriminatory interactions in unique ways (23), and as a consequence, instances have been reported in which aptamers did not bind denatured target proteins (24–26).…”