IDEAL-FILTER CAPILLARY ELECTROPHORESIS (IFCE) AS A UNIVERSAL METHOD FOR SELECTION OF DNA APTAMERS FROM OLIGONUCLEOTIDE LIBRARIES

Svetlana Krylova, Chemistrty, York University, Toronto, Canada (krylova@yorku.ca)
An T., Chemistrty, York University, Toronto, Canada
Sergey N., Chemistrty, York University, Toronto, Canada

Aptamers revolutionized many areas of analytical and medical chemistry ranging from diagnostics of diseases to drug development. One of the popular methods of selection of DNA aptamers from random-sequence DNA libraries is non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM), which offers a simple and rapid separation of protein–DNA complexes from the unbound DNA molecules in an electric field. However, sometimes we find that while the majority of DNA molecules migrate with predictable velocities, there is a fraction of DNA molecules that migrates with a wide range of irregular velocities. This irregular fraction of DNA can be quantitated by qPCR detection. We proved that the interaction between DNA and its condensed counter ions caused this non-uniform migration of DNA in electric field. Since protein targets often require relatively high salt concentrations, selection of DNA aptamers in low salt conditions is not desirable. The study of DNA velocity profiles in solutions containing physiological levels of salt was critical for selection of aptamers. To resolve a problem of the presence of irregular fraction of DNA we suggested moving protein–DNA complexes and the unbound DNA in the opposite directions in electric field in an uncoated capillary. This idea led us to the development of Ideal-Filter Capillary Electrophoresis (IFCE) as a uniquely efficient method for selection of affinity ligands from oligonucleotide libraries, such as random-sequence RNA and DNA libraries as well as DNA-encoded libraries (DELs)*. In this approach, the efficiency of partitioning was optimized by changing the running buffer’s ionic strength to reach a value of 10⁹. This value is 10⁴ times higher than the highest previously reported value for NECEEM and 10⁷ times higher that the best values achievable with practical solid-phase partitioning methods (e.g. with a target protein immobilized on magnetic beads) used in aptamer selection.

* A. T. H. Le, S. M. Krylova, M. Kanoatov, S. Desai, S. N. Krylov. Angew. Chem. Int. Ed. 2019, 58, 2739 –2743.

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Abstract Reference & Short Personal Biography of Presenting Author

Dr. Krylova obtained her PhD from the Russian Academy of Sciences. She has over ten years of research leadership experience in the area of medical diagnostics and drug development in biotechnology and pharmaceutical companies in Canada. She has been a contract faculty member at York University in Toronto since 2008. Dr. Krylova is also leading research projects in the area of Bioanalytical Chemistry as a Senior Research Associate in the Centre for Research on Biomolecular Interactions at York University.