Analytical Chemistry

Quantitative Characterization of Partitioning in Selection of DNA Aptamers for Protein Targets by Capillary Electrophoresis



Partitioning of protein–DNA complexes from protein-unbound DNA is a key step in selection of DNA aptamers. Conceptually, the partitioning step is characterized by two parameters: transmittance for protein-bound DNA (binders) and transmittance for unbound DNA (nonbinders). Here we present the first study to reveal how these transmittances depend on experimental conditions; such studies are pivotal to the effective planning and control of selection. Our focus was capillary electrophoresis (CE) which is a partitioning approach of high efficiency. By combining a theoretical model and experimental data, we evaluated the dependence of transmittances of binders and nonbinders on the molecular weight of protein target in two modes of CE-based partitioning: nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) and ideal-filter capillary electrophoresis (IFCE). Our data suggest that as the molecular weight of the protein target decreases: (i) the transmittance for binders remains close to unity in NECEEM but decreases drastically in IFCE and (ii) the transmittance for nonbinders increases orders of magnitude in NECEEM but remains relatively stable at a very low level in IFCE. To determine the optimal CE conditions for a given size of protein target, a balance between transmittances of binders and nonbinder must be reached; such a balance would ensure the collection of binders of sufficient purity and quantity. We conclude that, as a rule of thumb, IFCE is preferable for large-size protein targets while NECEEM should be the method of choice for small-size protein targets


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Supplementary material

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Data and calculations for figure preparation
This excel file contains the raw data and calculations used to plot all the figures in the manuscript and supporting information, including: 1) the calculation of predicted mobility and elution time of protein–DNA complex for 3 types of CE-based partitioning, 2) raw data of background-measurement experiments: number of DNA molecules versus elution time, 3) calculation of predicted binder-collection windows and the associated kN values, 4) calculation of predicted kB values with 2 values of koff = 10^−3 and 10^−4 and 5) calculation of required (mmin) and allowed (mmax) numbers of partitioning rounds in non-SELEX selection of aptamers.