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

Abstract

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 the 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 nonbinders 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 largesize protein targets while NECEEM should be the method of choice for small-size protein targets.

Transient Incomplete Separation of Species with Close Diffusivity to Study the Stability of Affinity Complexes

“…36 NECEEM is a well-established method with wellunderstood limitations. 37 The quality of separation in NECEEM is, in general, better than in ACTIS (NECEEM can provide baseline separation of the unbound aptamer from the protein-bound aptamer). On the downside, NECEEM separation is much longer (∼10 min) than ACTIS separation (<1 min), and accordingly much more prone to the adsorption of T, L, and TL onto the capillary walls.…”

“…While ACTIS has been proven to be intrinsically accurate, we decided to confirm its result by using another solution-based method: NECEEM . NECEEM is a well-established method with well-understood limitations . The quality of separation in NECEEM is, in general, better than in ACTIS (NECEEM can provide baseline separation of the unbound aptamer from the protein-bound aptamer).…”

Bulk Affinity Assays in Aptamer Selection: Challenges, Theory, and Workflow

“…27 Because PVA coating suppresses the electroosmotic flow, we applied the "complex-last" NECEEM mode for the aptamer selection. 28 It is beneficial for affinity assays to use the lowest library concentration at which the signal-to-noise ratio (S/N) is still sufficiently high to ensure accurate R determination. To satisfy this condition, we chose [L] 0 = 1 nM for all of our bulk affinity assays.…”

“…We found that using PVA-coated capillaries can largely reduce the protein adsorption to capillary walls . Because PVA coating suppresses the electroosmotic flow, we applied the “complex-last” NECEEM mode for the aptamer selection …”

Systematic bio-fabrication of aptamers and their applications in engineering biology

“…Compared with the above two Mag SELEX methods that immobilize nucleic acid library or targets, CE SELEX has a higher resolution and faster target separation rate. Free targets, nucleic acids and nucleic acid-target complexes have different migration abilities in electrophoresis, thus they can be quickly separated by capillary electrophoresis, improving screening efficiency and accuracy [ 34 , 35 ]. At the same time, it can also avoid the contamination of the aptamer candidate library caused by the nucleic acid residue during the elution process.…”