Toward Automated Nucleic Acid Enzyme Selection
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L.J. Sooter
Abstract
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 beadbound 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.
Copyright © 2001 by Walter de Gruyter GmbH & Co. KG
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Articles in the same Issue
- Highlight: Evolution in Vivo, in Vitro and in Machina
- Modeling Genetic Networks and Their Evolution: A Complex Dynamical Systems Perspective
- Evolution in Silico and in Vitro: The RNA Model
- Divergent Evolution of (??)8-Barrel Enzymes
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- Toward Automated Nucleic Acid Enzyme Selection
- Duocalins: Engineered Ligand-Binding Proteins with Dual Specificity Derived from the Lipocalin Fold
- The Stochastic Evolution of Catalysts in Spatially Resolved Molecular Systems
- Fragment-Based Flexible Ligand Docking by Evolutionary Optimization
- Specific Nucleoprotein Complexes within Adenovirus Capsids
- ERH (Enhancer of Rudimentary Homologue), a Conserved Factor Identical between Frog and Human, Is a Transcriptional Repressor
- Signal Transduction by the Chemokine Receptor CXCR5: Structural Requirements for G Protein Activation Analyzed by Chimeric CXCR1/CXCR5 Molecules
- Arginine-Specific Cysteine Proteinase from Porphyromonas gingivalis as a Convenient Tool in Protein Chemistry
- Chemokine-Induced Secretion of Gelatinase B in Primary Human Monocytes
