Abstract
Background: The apply of aptamers as a new generation’s way to probe diagnostic for the detection of target molecules has gained ground. Aptamers can be used as alternatives to diagnostic antibodies for detection of blood groups due to their unique features. This study was aimed to produce DNA diagnostic aptamer detecting the antigen of A1 blood group using the Cell-Selex method. Materials and Methods: DNA aptamer was isolated against A1 RBC antigen after ten stages of Cell-Selex and amplification by an asymmetric polymerase chain reaction. The progress of the stages of selection was evaluated using flow cytometry analysis, which the DNA aptamer isolated from the tenth cycle with an affinity of 70% fluorescent intensity, was selected from four positive colonies followed by determination of the sequences and secondary structures. Results: The aptameric sequence obtained from C4 cloning was calculated with the highest binding affinity to A1 antigen having an apparent dissociation constant (Kd value) of at least 29.5 ± 4.3 Pmol, which was introduced as the selected aptamer-based on ΔG obtained from a colony of C4 equal to –13.13. Conclusion: The aptamer obtained from using Cell-Selex method could be used as an example for the development of diagnostic tools such as biosensors for detecting A1 blood group antigens.
Highlights
One of the first and most basic tests in transfusion bank is to determine the grouping of antigens on red blood cell (RBC) surface based on the ABO blood group system
The aptamers target nearby molecules with pre-designed specific primers by a mode of selection called systematic evolution of ligands by exponential enrichment (SELEX), and they are amplified by polymerase chain reaction (PCR) through washing process [8]
Selex cycles are sequentially repeated between 6 to 20 times to yield a nucleic acid ligand with the highest affinity to the target molecule, after which the process ceases, the selection and progress processes are monitored by screening tools like flow cytometry and ELISA to achieve the highest affinity [10, 11]
Summary
One of the first and most basic tests in transfusion bank is to determine the grouping of antigens on red blood cell (RBC) surface based on the ABO blood group system. Aptamers are short-chain, single-stranded DNA or RNA with a length of 90–100 nucleotides They can bind to targets including protein, peptides, amino acids, ATP, antibodies, small chemical molecules, viruses, whole cells, cellular receptors, antigens, metal ions, and enzymes based on their complex 3-D constructs. This study aimed to identify and locate DNA aptamers with a high specific affinity to A1 RBC using Cell-Selex approach and asymmetric amplification, which selected aptamers were confirmed by flow cytometry analysis followed by sequencing and prediction of their secondary structures that can replace antibodies. This study was aimed to produce DNA diagnostic aptamer detecting the antigen of A1 blood group using the Cell-Selex method. Conclusion: The aptamer obtained from using Cell-Selex method could be used as an example for the development of diagnostic tools such as biosensors for detecting A1 blood group antigens. [GMJ.2020;9:e1657] DOI:10.31661/gmj.v9i0.1657
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