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 workstation. Several binding species with nanomolar affinities were isolated from diverse populations. Automation of a deoxyribozyme ligase selection is 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. Initially, 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, pipetting 50% glycerol solutions, purifying single strand DNA, and monitoring the progress of the selection as it is performed. Nonetheless, automated selection experiments for deoxyribozyme ligases were carried out starting from either a naive pool or round eight of the manually selected pool. In both instances, the first round of selection revealed an increase in ligase activity. However, this activity was lost in subsequent rounds. A possible cause could be mispriming during the unmonitored PCR reactions. Potential solutions include pool redesign, fewer PCR cycles, and integration of a fluorescence microtiter plate reader to allow robotic 'observation' of the selections as they progress.
Sooter
L J
LJ
Department of Chemistry and Biochemistry, Institute for Cell and Molecular Biology, University of Texas at Austin, 78712, USA.
Riedel
T
T
Davidson
E A
EA
Levy
M
M
Cox
J C
JC
Ellington
A D
AD
D001331:Automation; D001483:Base Sequence; D021881:DNA, Catalytic / Q000378:metabolism*; D008025:Ligases / Q000737:chemistry* / Q000378:metabolism; D009696:Nucleic Acids / Q000737:chemistry*
D021881:DNA, Catalytic; D009696:Nucleic Acids; D008025:Ligases