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Three-dimensional selection of leptin aptamers using capillary electrophoresis and implications for clone validation
1Department of Chemistry, National University of Singapore, Singapore 117543, Singapore.
Analytical Biochemistry
|December 13, 2012
Summary
Capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX) identified aptamers for human leptin. Validation methods yielded different binding affinities, highlighting the need for consistent assay environments.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Molecular Biology
Background:
- Capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX) is a method for aptamer selection against protein targets.
- CE-SELEX offers advantages in separating bound and unbound DNA in a solution environment.
Purpose of the Study:
- To select aptamers against human leptin protein using CE-SELEX.
- To compare different methods for validating aptamer binding affinity.
Main Methods:
- Capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX) was employed for aptamer selection.
- Aptamer binding affinity was validated using Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) and a fluorescence intensity method.
Main Results:
- CE-SELEX successfully selected aptamers against human leptin.
- NECEEM yielded dissociation constants (K(D)) in the high nanomolar range.
- The fluorescence intensity method resulted in K(D) values in the low micromolar range.
Conclusions:
- Aptamer validation environments should mimic selection and intended use conditions.
- Fluorescence intensity methods may be limited by target dissociation rates (k(off)).
- Discrepancies in validation results can arise from differing techniques and target immobilization effects.
