Novel Approach to Overcome Defects of Cell-SELEX in Developing Aptamers against Aspartate β-Hydroxylase

Abstract

Cell-based aptamer selection (Cell-SELEX) against predefined protein targets that benefits using the native form of the targets is the most promising approach to achieve aptamer probes capable of recognizing targets under both in vitro and in vivo conditions. The major disadvantages in Cell-SELEX are the imperfectness of the negative selection step and the lengthy procedure of selection. Here, we introduced the Counter-SELEX as part of our modified Cell-SELEX and implemented deep sequencing to overcome these shortcomings in developing aptamers against aspartate β-hydroxylase (ASPH) as a known tumor marker. In parallel with the conventional Cell-SELEX, five consecutive cycles of counter selection were accomplished using sequences bound to negative cells (the Counter-SELEX) to detect oligos that are not specific for ASPH. After high-throughput sequencing, the representative of each promising achieved family was subjected to further confirmatory analysis via flow cytometry, followed by the fluorescence immunostaining of histopathological sections. Implementing our innovative complementary method, annoying mis-selected sequences in Cell-SELEX enriched pools were effectively identified and removed. According to the affinity assay on the cells displaying ASPH, three aptamers, AP-Cell 1, AP-Cell 2, and AP-Cell 3, with K d values of 47.51, 39.38, and 65.23 nM, respectively, were obtained, while AP-Cell 1 and 3 could then successfully spot ASPH displayed on the tissues. Our study showed that the Counter-SELEX could be considered as a complementary method for Cell-SELEX to overcome the imperfectness of the negative selection step. Moreover, high-throughput nucleotide sequencing could help to shorten the overall process.

Identification of G-quadruplex anti-Interleukin-2 aptamer with high specificity through SELEX stringency

“…Moreover, the production of aptamers could be performed against a lower amount of target protein (in micrograms) in comparison with antibodies, which need an amount of large protein (in milligrams) [ 10 ]. Aptamer production is carried out in a variety of processes with SELEX techniques against proteins [ 19 ] and peptides [ 20 ] and with the Cell-SELEX technique against cell surface receptors or antigens [ 21 , 22 , 23 , 24 ].…”

New Insights into Aptamers: An Alternative to Antibodies in the Detection of Molecular Biomarkers

“…Using this approach, the Cell-SELEX protocol was successfully used to generate aptamers that can specifically target cancer cells, which involves positive and negative selection. A negative selection step is necessary to remove sequences that bind to normal cells and to improve the specificity of aptamer candidates [68]. The SELEX process was modified to Cell-SELEX in order to identify aptamers for pathogens such as Streptococcus, Listeria monocytogenes, Salmonella, Staphylococcus aureus, Vibrio parahaemolyticus, Escherichia coli, and Pseudomonas aeruginosa [69].…”

Computational Frontiers in Aptamer-Based Nanomedicine for Precision Therapeutics: A Comprehensive Review

“…For instance, immunoprecipitation-coupled SELEX was created to increase affinity in physiological settings, 28 whereas capture-SELEX was planned to increase aptamer selection efficiency for unidentified short targets of molecular biology. 29 Furthermore, complete cells, 30 tissue, or organs in animals 31 have been employed as SELEX tools for discriminating between healthy and pathological situations, 32 monitoring prognosis, 33 and identifying novel biomarkers. 34 Emerging of advanced technologies, like microfluidics, 35 capillary electrophoresis, 36 atomic force microscopy, 37 and numerous in-silico methods, were additionally employed to improve the SELEX strategy’s effectiveness at this stage.…”