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Selection of bovine catalase aptamers using non‐ SELEX
Author(s) -
Ashley Jon,
Ji Kaili,
Li Sam F. Y.
Publication year - 2012
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201200032
Subject(s) - aptamer , systematic evolution of ligands by exponential enrichment , capillary electrophoresis , trypsinogen , chemistry , chromatography , biosensor , myoglobin , lysozyme , affinity electrophoresis , affinity chromatography , microbiology and biotechnology , biochemistry , trypsin , biology , rna , enzyme , gene
In this research, we used the non‐ SELEX method to successfully select an aptamer that binds to the protein target (bovine catalase) with a K D value in the low micro molar range. The time window was determined for the target and aptamer library by optimizing the buffer conditions using 3 × T ris‐glycine‐potassium phosphate ( TGK ) buffer as the run buffer and 1× TGK as the selection buffer to give the biggest complex peak. Fractions were collected by multistep nonequilibrium capillary electrophoresis of equilibrium mixtures ( NECEEM )‐based partitioning for three rounds of selection. The fractions from each round were enriched using PCR and the progress of selection was monitored using bulk affinity analysis. Fraction 2 was determined to have the optimal bulk affinity (0.75 μM) and this enriched library was cloned and sequenced giving four aptamer sequences. These sequences were verified using affinity capillary electrophoresis ( CAT 1 0.237 μM) and fluorescence intensity measurements ( CAT 1 0.430 μM). The specificity of the aptamer was also determined by fluorescence intensity measurements. The results showed that the aptamer with the highest binding affinity showed at least a 100‐fold decrease in affinity toward four other proteins (i.e. lysozyme, trypsinogen, chymotrypsinogen A , and myoglobin) tested and this confirmed that the aptamer exhibited a distinct specificity toward bovine catalase. This aptamer will be useful in biosensing, W estern blot, and biomarker identification.