Isolation of aptamers with binding affinity for reduced and oxidized glutathione

Single stranded oligonucleotides called aptamers that have a binding affinity to reduced glutathione (GSH) and oxidized glutathione (GSSG) were isolated. An iterative technique called Systematic Evolution of Ligands by Exponential Enrichment (SELEX) was used to obtain an enriched aptamer pool of randomized sequences for GSH and another enriched aptamer pool for GSSG. Quantification via UV-visible spectroscopy yielded an aptamer pool with a nucleic acid concentration of 610.4 ng/L for GSH and 660.4 ng/L for GSSG. The resulting amplicons were inserted into E. coli for bacterial cloning to allow for DNA extraction and sequencing. Twenty-four GSH-binding aptamers and twenty-four GSSG-binding aptamers were isolated and sequenced. The GSH-binding aptamers are sensitive to as low as a 0.1 mM GSH solution while the GSSG-binding aptamers are sensitive to as low as a 0.01 mM GSSG solution. The Unified Nucleic Acid Folding Mfold web server was used to predict aptamer structures. The aptamers were analyzed for consistent sequences and structural motifs. Future studies should focus on testing the obtained aptamers with fluorescence binding assay to determine the sequence and structural motif with the highest binding affinity to GSH and GSSG respectively.