JUN 02, 2016 08:00 AM PDT

WEBINAR: CE-SELEX: Isolating High Affinity Aptamers Using Capillary Electrophoresis

SPONSORED BY: Sciex, Sciex
C.E. CREDITS: P.A.C.E. CE
Speakers
  • Professor of Chemistry, University of Minnesota
    Biography
      Prof. Michael Bowser received his B.Sc. in Chemistry from Dalhousie University in 1994. He received his Ph.D. in Chemistry from the University of British Columbia in 1998 where, under the guidance of Prof. David Chen, he studied analyte-additive interactions and how they affect capillary electrophoresis separations. Prof. Bowser then moved to the University of Florida to continue his studies as an NSERC Postdoctoral Fellow in Prof. Robert Kennedy's group where he studied high-speed capillary electrophoresis separations and how they could be applied to the online analysis of neurotransmitter dynamics. In 2000, Prof. Bowser accepted a position at the University of Minnesota where is currently a Professor of Chemistry. The focus of the Bowser group is to advance bioanalytical technologies used to study problems of biological and medical importance. Major contributions include: 1) development of microfluidic strategies for isolating aptamers (i.e. CE-SELEX), 2) the design and characterization of microscale free flow electrophoresis devices for high-speed monitoring and microscale purification and 3) introduction of in vitro microdialysis, a technique that allows high-speed measurements of chemical release/uptake in cellular systems. Applications of particular interest include adipocyte signaling in obesity, aptamers that facilitate cell delivery, and high-speed multidimensional separations.

    Abstract:
         SELEX is a process that selects DNA or RNA from a random library of sequences based on their affinity for a target molecule.  These high affinity ligands, called aptamers, have great potential for use as drugs or diagnostic agents.  Traditionally, SELEX selection is performed using filters, panning or affinity chromatography separations and there are drawbacks to these traditional approaches.  They often require 8-15 selection rounds before a significant fraction of the pool shows affinity for the target and they can create the possibility of selecting for non-specific binders with affinity for the stationary support.
         This presentation focuses on a SELEX procedure that uses capillary electrophoresis to perform selections (CE-SELEX).  In this procedure a random sequence DNA library is incubated with the target in an injection vial.  The mixture is injected onto the capillary and a separation voltage is applied.  The size or charge of DNA with affinity for the target changes upon binding the target, inducing a mobility shift.  This mobility shift allows binding and non-binding sequences to be collected into separate vials.  Binding sequences are PCR amplified generating a new pool for further rounds of enrichment.  
         Highly selective aptamers have been identified with dissociation constants in the low nanomolar to high picomolar range for large protein targets such as IgE and HIV-RT.  More recently, aptamers have been identified with affinity for smaller targets such as neuropeptide Y and N-methylmesoporphyrin.  An important advantage of CE-SELEX results from the high efficiency, high selectivity separations characteristic of CE.  This increased separation power enhances the rate of enrichment between rounds, greatly shortening the SELEX process.  Selection takes place in free solution, minimizing non-specific interactions, increasing the abundance of high affinity aptamers in the final DNA pool.

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