title: A combinatorial systematic evolution of ligands by exponential enrichment method for selection of aptamer against protein targets.
abstract: Aptamers are synthetic DNA recognition elements which form unique conformations that enable them to bind specifically to their targets. In the present study, an attempt was made to standardize a new modified combinatorial method comprising of Ni-NTA affinity Systematic Evolution of Ligands by Exponential Enrichment (SELEX; based on affinity between His tag protein and Ni-NTA), membrane SELEX (based on immobilization of protein on nitrocellulose membrane), and microtiter plate based SELEX (to monitor affinity and to enrich the selected aptamers) for protein targets. For experimental evaluation, staphylococcal interotoxin B was the molecule chosen. The new combinatorial method enhanced selection ability up to 51.20 % in comparison with individual conventional procedures. Employing this method following six rounds of selection, high-affinity aptamers with very different properties could be obtained with a dissociation constant (K d) value as low as 34.72 ± 25.09 nM. The optimal aptamers could be employed in fluorescence binding assay, enzyme-linked oligonucleotide assays, and aptamer-based Western blot assay for characterization and detection. These results pave a potential path without using of any robotics for high-throughput generation of aptamers with advantages in terms of rapidity, simplicity, and ease in handling.
authors: Defence Food Research Laboratory (DFRL), Mysore, 570011, India. bhairabmicro21@gmail.com.
authors: Bhairab
authors: B
authors: Mondal
authors: Defence Food Research Laboratory (DFRL), Mysore, 570011, India. shylajaramlal1@gmail.com.
authors: Shylaja
authors: S
authors: Ramlal
authors: Defence Food Research Laboratory (DFRL), Mysore, 570011, India. sudhalavu1@gmail.com.
authors: Padma Sudha Rani
authors: PS
authors: Lavu
authors: Defence Food Research Laboratory (DFRL), Mysore, 570011, India. drmuralihs@yahoo.co.in.
authors: Harishchandra Sreepathy
authors: HS
authors: Murali
authors: Defence Food Research Laboratory (DFRL), Mysore, 570011, India. drharshavardanbatra@gmail.com.
authors: Harsh Vardhan
authors: HV
authors: Batra
mesh_terms: D052157:Aptamers, Nucleotide / Q000302:isolation & purification* / Q000378:metabolism*; D004768:Enterotoxins / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D052156:SELEX Aptamer Technique / Q000379:methods*
chemical_list: D052157:Aptamers, Nucleotide; D004768:Enterotoxins; C031818:enterotoxin B, staphylococcal
keywords: Asymmetric polymerase chain reaction; Combinatorial systematic evolution of ligands by exponential enrichment; DNA aptamer; Enterotoxin B; Enzyme-linked oligonucleotide assay