In vitro evolution of functional DNA using capillary electrophoresis.
Electrophoretic selection with capillary electrophoresis (CE) is used, for the first time, to isolate functional nucleic acid sequences using SELEX (systematic evolution of ligands by exponential enrichment). SELEX uses molecular evolution to select functional sequences (aptamers) from random RNA or DNA libraries. Conventional SELEX is usually performed with affinity chromatography, which may introduce significant bias into the selection step. Important biases include the slow kinetics involved in the elution of strongly bound sequences and performing the selection with the target molecule tethered to the stationary support, not in free solution. In this novel CE-SELEX approach, selection occurs in free solution. The nucleic acid sequences that bind the target undergo a mobility shift, migrating at a different rate, allowing them to be separated from the inactive sequences. Thus, there is no need to wash the active sequences off a column as in conventional SELEX, eliminating any kinetic bias. In this work, the viability of CE-SELEX was demonstrated by performing selections against immunoglobulin E (IgE). Anti-IgE aptamers with dissociation constants as low as 40 nM were obtained in only two rounds of selection.
Mendonsa
Shaun D
SD
Department of Chemistry, University of Minnesota, 207 Pleasant Street SE, Minneapolis, Minnesota 55455, USA.
Bowser
Michael T
MT
D000818:Animals; D004247:DNA / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D007073:Immunoglobulin E / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D007700:Kinetics; D051379:Mice
D007073:Immunoglobulin E; D004247:DNA