Nonequilibrium Capillary Electrophoresis of Equilibrium Mixtures: A Universal Tool for Development of Aptamers

Aptamers are DNA (or RNA) ligands selected from large libraries of random DNA sequences and capable of binding different classes of targets with high affinity and selectivity. Both the chances for the aptamer to be selected and the quality of the selected aptamer are largely dependent on the method...

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Bibliographic Details
Published in:Journal of the American Chemical Society 2005-03, Vol.127 (9), p.3165-3171
Main Authors: Berezovski, Maxim, Drabovich, Andrei, Krylova, Svetlana M, Musheev, Michael, Okhonin, Victor, Petrov, Alexander, Krylov, Sergey N
Format: Article
Language:English
Subjects:
Alkyl and Aryl Transferases - chemistry
Alkyl and Aryl Transferases - genetics
Alkyl and Aryl Transferases - metabolism
Analytical, structural and metabolic biochemistry
Base Sequence
Biological and medical sciences
DNA - chemistry
DNA - genetics
DNA - metabolism
Electrophoresis, Capillary - methods
Farnesyltranstransferase
Fundamental and applied biological sciences. Psychology
Gene Library
General aspects, investigation methods
Kinetics
Nucleic acids
Oligonucleotides - chemistry
RNA - chemistry
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Summary:Aptamers are DNA (or RNA) ligands selected from large libraries of random DNA sequences and capable of binding different classes of targets with high affinity and selectivity. Both the chances for the aptamer to be selected and the quality of the selected aptamer are largely dependent on the method of selection. Here we introduce selection of aptamers by nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM). The new method has a number of advantages over conventional approaches. First, NECEEM-based selection has exceptionally high efficiency, which allows aptamer development with fewer rounds of selection. Second, NECEEM can be equally used for selecting aptamers and finding their binding parameters. Finally, due to its comprehensive kinetic capabilities, the new method can potentially facilitate selection of aptamers with predefined K d, k off, and k on of the aptamer−target interaction. In this proof-of-principle work, we describe the theoretical bases of the method and demonstrate its application to a one-step selection of DNA aptamers with nanomolar affinity for protein farnesyltransferase.
ISSN:0002-7863
1520-5126
DOI:10.1021/ja042394q