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Abstract
We report here the in vitro selection of DNA aptamers for electric eel acetylcholinesterase (AChE). One selected aptamer sequence (R15/19) has a high affinity towards the enzyme (Kd = 157 ± 42 pM). Characterization of the aptamer showed its binding is not affected by low ionic strength (~20 mM), however significant reduction in affinity occurred at high ionic strength (~1.2 M). In addition, this aptamer does not inhibit the catalytic activity of AChE that we exploit through immobilization of the DNA on a streptavidin-coated surface. Subsequent immobilization of AChE by the aptamer results in a 4-fold higher catalytic activity when compared to adsorption directly on to plastic.
Highlights
In vitro selection of nucleic acid aptamers has become increasingly used to create new tailored molecules of interest for a variety of applications
We present the use of an aptamer as a surface bound immobilization reagent
The results show the aptamer binds AChE with high affinity (Kd = 157 ± 42 pM) whilst no binding to bovine serum albumin (BSA) or streptavidin was detected
Summary
In vitro selection of nucleic acid aptamers has become increasingly used to create new tailored molecules of interest for a variety of applications. Efforts to avoid this involve site-directed immobilization, commonly by the addition of affinity tags via recombinant DNA methods, which has become popular due to its one-step attachment strategy that immobilizes the enzyme molecules in a site-directed and oriented fashion and facilitates substrate access [14] Such affinity tags include Biotin, His-tag, the FLAG octapeptide, Strep-II, glutathione S-transferase (GST), maltose binding protein (MBP), and heavy chain protein-C (HPC) which have been shown to be able to efficiently hold the enzymes without significantly disturbing their function [15,16,17,18].
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