Development and characterization of DNA aptamer against Retinoblastoma by Cell-SELEX

Abstract

Retinoblastoma (RB) is the most common paediatric intraocular tumour. The management of RB has improved the survival and vision with recent advances in the treatment. Improved therapeutic approaches focussing on targeting tumours and minimizing the treatment-associated side effects are being developed. In this study, we generated a ssDNA aptamer against RB by cell-SELEX and high-throughput sequencing using Weri-RB1 cell line as the target, and Muller glial cell line Mio-M1 as the control. Three aptamers were selected based on the number of repetitions in NGS and phylogenetic relationship and evaluated by flow cytometry to assess their binding affinity and selectivity. The dissociation constant, Kd values of three selected aptamers were found to be in the nanomolar range. Aptamer VRF-CSRB-01 with the best binding affinity and a Kd value of 49.41 ± 7.87 nM was further characterized. The proteinase and temperature treatment indicated that VRF-CSRB-01 targets surface proteins, and has a good binding affinity and excellent selectivity under physiological conditions. The aptamer VRF-CSRB-01 was stable over 72 h in serum and 96 h in cerebral spinal fluid and vitreous. With the high affinity, specificity, stability and specific recognition of clinical RB tumours, VRF-CSRB-01 aptamer holds potential for application in diagnosis and targeting RB.

Development of DNA aptamers targeting B7H3 by hybrid-SELEX: an alternative to antibodies for immuno-assays

“…During each cycle of protein-SELEX, PCR-amplified dsDNA was identified and passed through the affinity purification on streptavidin-sepharose beads followed by alkaline separation to get FITC-labelled ssDNA, which was used as an input for the sequential cycle of cell-SELEX. Cell-SELEX was performed using Weri-RB1 cells for positive selection and Mio-M1 for negative selection as described in Maradani et al 14 . The enriched ssDNA of the cell-SELEX cycle was used as input for the next cycle of protein-SELEX of successive round of the selection.…”

“…The initial library was selected by screening the binding affinities of enriched pools of RB cell-SELEX (Maradani et al 14 ) on recombinant B7H3 protein by dot-blot. Briefly 10 ng of the recombinant B7H3 was blotted on to the methanol activated PVDF membrane and allowed to bind.…”

“…The mean fluorescence intensity of each sample was subtracted from the mean fluorescence intensity of the background produced by the ssDNA library. The equilibrium dissociation constant (Kd) of the aptamer–cell interaction was obtained using GraphPad Prism (GraphPad Prism version 7.00 for Windows) by fitting the dependence of fluorescence intensity of specific binding on the concentration of the aptamers to the equation: where Y is fluorescence intensity of cells, X is concentration of the aptamer and B max is maximum binding potential 14 .…”

“…The FFPE sections were deparaffinized and antigen retrieval was done by a standard procedure using 0.01 M citrate buffer (pH 6.0). IHC with aptamers was carried out as mentioned in Maradani et al 14 . For IHC with the antibody, antigen-retrieved sections were blocked with HRP blocker (Dako K8023) and washed three times with PBS.…”

“…Several studies have compared aptamers to antibodies to detect proteins in immuno-assays. Aptamers have replaced antibodies in histochemical staining of both fresh-frozen and paraffin-embedded tissue sections 11 – 14 , flow-cytometry 14 – 17 , dot-blot 18 , 19 , western blot 20 , 21 , ELISA 22 , 23 and many other antibody-dependent assays. All the above studies are about the application of aptamers in comparison antibodies to a single assay.…”