Toward automated nucleic acid enzyme selection

L. J. Sooter, T. Riedel, E. A. Davidson, M. Levy, J. C. Cox, A. D. Ellington

Research output: Contribution to journalArticle

23 Citations (Scopus)

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 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.

Original languageEnglish (US)
Pages (from-to)1327-1334
Number of pages8
JournalBiological Chemistry
Volume382
Issue number9
DOIs
StatePublished - 2001
Externally publishedYes

Fingerprint

Catalytic DNA
Ligases
Nucleic Acids
Automation
Robotics
Enzymes
DNA
Polymerase Chain Reaction
Washing
Iodine
Glycerol
Ligation
Fluorescence
Observation
Catalysts
Monitoring
Substrates
Population
Experiments

Keywords

  • Deoxyribozyme
  • In vitro selection
  • Ligase
  • Robot
  • SELEX

ASJC Scopus subject areas

  • Biochemistry

Cite this

Sooter, L. J., Riedel, T., Davidson, E. A., Levy, M., Cox, J. C., & Ellington, A. D. (2001). Toward automated nucleic acid enzyme selection. Biological Chemistry, 382(9), 1327-1334. https://doi.org/10.1515/BC.2001.165

Toward automated nucleic acid enzyme selection. / Sooter, L. J.; Riedel, T.; Davidson, E. A.; Levy, M.; Cox, J. C.; Ellington, A. D.

In: Biological Chemistry, Vol. 382, No. 9, 2001, p. 1327-1334.

Research output: Contribution to journalArticle

Sooter, LJ, Riedel, T, Davidson, EA, Levy, M, Cox, JC & Ellington, AD 2001, 'Toward automated nucleic acid enzyme selection', Biological Chemistry, vol. 382, no. 9, pp. 1327-1334. https://doi.org/10.1515/BC.2001.165
Sooter LJ, Riedel T, Davidson EA, Levy M, Cox JC, Ellington AD. Toward automated nucleic acid enzyme selection. Biological Chemistry. 2001;382(9):1327-1334. https://doi.org/10.1515/BC.2001.165
Sooter, L. J. ; Riedel, T. ; Davidson, E. A. ; Levy, M. ; Cox, J. C. ; Ellington, A. D. / Toward automated nucleic acid enzyme selection. In: Biological Chemistry. 2001 ; Vol. 382, No. 9. pp. 1327-1334.
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