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Generation of HBsAg DNA aptamer using modified cell-based SELEX strategy

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Abstract

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.

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Acknowledgements

We are grateful to the Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.

Funding

This work was financially supported by Isfahan University of Medical Sciences (Grant No. 394169).

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Authors and Affiliations

  1. Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Science, Isfahan University of Medical Sciences, Isfahan, Islamic Republic of Iran

    Mina Mirian & Ali Jahanian-Najafabadi

  2. Isfahan Neurosciences Research Center, Alzahra Research Institute, Isfahan University of Medical Sciences, Isfahan, Islamic Republic of Iran

    Shirin Kouhpayeh

  3. Applied Physiology Research Center, Cardiovascular Research Institute, Isfahan University of Medical Sciences, Isfahan, Islamic Republic of Iran

    Laleh Shariati

  4. Department of Biomaterials, Nanotechnology and Tissue Engineering, School of Advanced Technologies in Medicine, Isfahan University of Medical Sciences, Isfahan, Islamic Republic of Iran

    Laleh Shariati & Maryam Boshtam

  5. Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Islamic Republic of Iran

    Ilnaz Rahimmanesh, Leila Darzi, Razieh Taghizadeh & Hossein Khanahmad

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  1. Mina Mirian
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  2. Shirin Kouhpayeh
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Correspondence to Hossein Khanahmad.

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Fig. S1

PCR optimization. Agarose electrophoresis gel images of preparative PCR showing the products of the various cycles of selected DNA library amplification for 2 nd and 6th rounds of selection. The best number of cycles without any unspecific bands for the 2 nd and 6th rounds of selection are 16, and 18 cycles respectively which are illustrated with white arrows. Lane 1: 50-bp DNA ladder. Lane 2: Negative control. Lane 3 to 8: 8, 10, 12, 14, 16 and 18 cycle of amplifications (TIF 183 kb)

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Mirian, M., Kouhpayeh, S., Shariati, L. et al. Generation of HBsAg DNA aptamer using modified cell-based SELEX strategy. Mol Biol Rep 48, 139–146 (2021). https://doi.org/10.1007/s11033-020-05995-2

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  • DOI: https://doi.org/10.1007/s11033-020-05995-2

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