Optimizing NECEEM-Based Aptamer Selection Using Emulsion PCR To Obtain Potential DNA Inhibitors For The Human Dealkylating Enzyme ABH2
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The oldest chemotherapeutic drugs still in use today are the alkylating agents. In many cases, their effectiveness is hindered by the ability of cancerous cells to develop resistance using the cell’s innate DNA repair enzymes, such as hABH2. Targeted inhibition of such enzymes can potentially greatly improve chemotherapy. Aptamers are more efficient, versatile and theoretically easier to discover than conventional small molecule inhibitors. However, Aptamer selection is regularly unsuccessful and screening inhibitors is a lengthily process. Here we optimize the aptamer selection process using Emulsion PCR to improve the amplification efficiency post NECEEM separation and rapidly select high affinity aptamers to unmodified hABH2 in 4 rounds. We then show the efficiency and robustness of the recently introduced direct CE-based approach to rapidly measure the demethylation activity of hABH2 and hABH3 towards ssDNA 3-meC substrates and discuss its potential as method to identify aptamers able to selectively inhibit hABH2.