Purpose: To select ssDNA aptamers with binding affinity for norovirus genogroup I (GI) strains using a novel Graphene Oxide (GO)-based SELEX (Systematic Evolution of Ligands by EXponential enrichment) method.
Methods: A pool of random ssDNA molecules was mixed with 100 µg GO, incubated, and the aptamer-bound GO harvested by centrifugation. The GO was then incubated with a cocktail of virus-like particles (VLPs) (representing GI.1, GI.4, GI.6, GI.7 and GI.8 strains), which acted as targets for desorption of the aptamer sequences having specific VLP binding affinity. The VLP-aptamer bound moieties, present in solution, were separated from the GO by centrifugation. The supernatant was recovered, aptamer sequences amplified by PCR, gel-purified, and the pool reprocessed for another selection round. After four rounds, enriched aptamer pools were cloned, sequenced and their secondary structure analyzed using DNA Mfold. Candidates were screened for binding affinity to 12 VLPs corresponding to GI and GII norovirus strains using an Enzyme-Linked Apatmer Sorbant Assay (ELASA).
Results: Six unique clones were obtained (AP1 through AP6) having predicted free energy (dG) (Kcal/mol) values in the range of -5.78 to -10.99. Combined sequence analysis revealed six common domains (A-F). AP4 had strong binding affinity to GI.1 and GII.2 VLPs (signal intensity ratios of 6.4±0.4 and 7.8±0.7, respectively).
Significance: GO-based SELEX is a simple platform for the isolation of aptamers without requiring target elution. To our knowledge, this is the first instance of specific GI norovirus aptamer selection using VLPs in cocktail. Future work will focus on using the aptamers in detection platforms for clinical and food/environmental sample matrices.