title: Emulsion PCR significantly improves nonequilibrium capillary electrophoresis of equilibrium mixtures-based aptamer selection: allowing for efficient and rapid selection of aptamer to unmodified ABH2 protein.
abstract: Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM), a homogeneous approach to select DNA aptamers, is among the most efficient partitioning techniques. In contrast with surface-based systematic evolution of ligands by exponential enrichment (SELEX) approaches, the ability of NECEEM to select aptamers to unmodified proteins in solution is preferable for identifying aptamers for eventual in vivo use. The high stringency and low sample volumes of NECEEM, although generally beneficial, can result in binding of very few aptamers, requiring highly efficient amplification to propagate them. When amplified with standard PCR, detectable library enrichment can fail due to the fast conversion of the aptamers into byproducts and preferential amplification of nonbinders. As an alternative, we proposed the use of emulsion PCR (ePCR), which is known to reduce byproduct formation, as a PCR mode for coupling with NECEEM partitioning. For the first time, we tested the advantages of ePCR in NECEEM-based aptamer selection to a medically relevant DNA repair enzyme, ABH2. We report that the combination of ePCR with NECEEM allowed for the selection of aptamers in the first three rounds of SELEX, while SELEX with conventional PCR failed in a number of attempts. Selected aptamers to an unmodified ABH2 protein have potential use in diagnostics and as leads for anticancer cotherapies, used as enhancements of alkylating agents in chemotherapy.
authors.affiliation: Department of Chemistry and Centre for Research on Biomolecular Interactions, York University , Toronto, Ontario M3J 1P3, Canada.
authors.forename: Roman
authors.initials: R
authors.lastname: Yufa
authors.forename: Svetlana M
authors.initials: SM
authors.lastname: Krylova
authors.forename: Christine
authors.initials: C
authors.lastname: Bruce
authors.forename: Eleanor A
authors.initials: EA
authors.lastname: Bagg
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Schofield
authors.forename: Sergey N
authors.initials: SN
authors.lastname: Krylov
mesh_terms: D000071499:AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase; D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D045643:DNA Repair Enzymes / Q000737:chemistry* / Q000235:genetics; D049308:Dioxygenases / Q000737:chemistry* / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods*; D004655:Emulsions / Q000737:chemistry*; D015723:Gene Library; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D052156:SELEX Aptamer Technique / Q000379:methods*
chemical_list: D052157:Aptamers, Nucleotide; D004655:Emulsions; D049308:Dioxygenases; C486440:ALKBH2 protein, human; D000071499:AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase; D045643:DNA Repair Enzymes