Toward automated nucleic acid enzyme selection

Methods for automation of nucleic acid selections are being developed. The selection of aptamers has been successfully automated using a Biomek 2000 w orkstation. Several binding species with nanomolar affinities were isolated from diverse populations. Automation of a deoxyribozyme ligase selection i s in progress. The process requires eleven times more robotic manipulations than an aptamer selection. The random sequence pool contained a 5' iodine residue and the ligation substrate contained a 3' phosphorothioate. Initial ly, a manual deoxyribozyme ligase selection was performed. Thirteen rounds of selection yielded ligators with a 400-fold increase in activity over the initial pool. Several difficulties were encountered during the automation of DNA catalyst selection, including effectively washing bead-bound DNA, pi petting 50% glycerol solutions, purifying single strand DNA, and monitoring the progress of the selection as it is performed. Nonetheless, automated s election experiments for deoxyribozyme ligases were carried out starting fr om either a naive pool or round eight of the manually selected pool. In bot h instances, the first round of selection revealed an increase in ligase ac tivity. However, this activity was lost in subsequent rounds. A possible ca use could be mispriming during the unmonitored PCR reactions. Potential sol utions include pool redesign, fewer PCR cycles, and integration of a fluore scence microtiter plate reader to allow robotic 'observation' of the select ions as they progress.