Generation of HBsAg DNA aptamer using modified cell-based SELEX strategy.
Mina MirianShirin KouhpayehLaleh ShariatiMaryam BoshtamIlnaz RahimmaneshLeila DarziRazieh TaghizadehAli Jahanian-NajafabadiHossein KhanahmadPublished in: Molecular biology reports (2021)
Aptamers as potential alternatives for antibodies could be employed against hepatitis B surface antigen (HBsAg), the great hallmark and first serological marker in HBV, for further theragnostic applications. Therefore, isolation HBsAg specific aptamer was performed in this study with a modified Cell-SELEX method. HEK293T overexpressing HBsAg and HEK293T as target and control cells respectively, were incubated with single-stranded rounds of DNA library during six SELEX and Counter SELEX rounds. Here, we introduced the new modified Cell-SELEX using deoxyribonuclease I digestion to separate single stranded DNA aptamers against the HBsAg. Characterization and evaluation of selected sequences were performed using flow cytometry analysis. The results led to isolation of 15 different ssDNA clones in six rounds of selection which were categorized to four clusters based on common structural motifs. The evaluation of SELEX progress showed growth in aptamer affinity with increasing in the cycle number. Taken together, the application of modified cell-SELEX demonstrated the isolation of HBsAg-specific ssDNA aptamers with proper affinity. Modified cell-SELEX as an efficient method can shorten the selection procedure and increase the success rate while the benefits of cell-based SELEX will be retained. Selected aptamers could be applied in purification columns, diagnostic kits, and drug delivery system against HBV-related liver cancer.
Keyphrases
- hepatitis b virus
- single cell
- cell therapy
- nucleic acid
- gold nanoparticles
- induced apoptosis
- bone marrow
- liver failure
- cell death
- flow cytometry
- signaling pathway
- cell free
- cell proliferation
- risk assessment
- minimally invasive
- endoplasmic reticulum stress
- climate change
- circulating tumor cells
- drug induced
- capillary electrophoresis
- magnetic nanoparticles