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

Abstract

Large molecules can be generically separated from small ones, though partially and temporarily, in a pressure-driven flow inside a capillary. This transient incomplete separation has been only applied to species with diffusion coefficients different by at least an order of magnitude. Here, we demonstrate, for the first time, the analytical utility of transient incomplete separation for species with close diffusion coefficients. First, we prove in silico that even a small difference in diffusivity can lead to detectable transient incomplete separation of species. Second, we use computer simulation to prove that such a separation can be used for the reliable determination of equilibrium dissociation constant (K d ) of complexes composed of similar-sized molecules. Finally, we demonstrate experimentally the use of this separation for the accurate determination of K d value for a protein−aptamer complex. We conclude that "accurate constant via transient incomplete separation" (ACTIS) can serve as a reference method for affinity characterization of protein−aptamer binding in solution.

Streamlined Data Processing for Determination of Equilibrium Dissociation Constants with Accurate Constant via Transient Incomplete Separation (ACTIS)

“…First, we showed that ACTIS is inherently accurate, and this accuracy is invariant to large variations in geometries of the fluidic system and the flow. , Second, this insensitivity to variations, in turn, allowed us to create a simple and robust fluidic system, which supports ACTIS accuracy . Third, we showed ACTIS to be applicable to protein–small molecule and protein–aptamer binding pairs. , Although ACTIS can potentially be used for all molecular complexes, including protein–protein binding pairs, future experimental work needs to be done to show such applicability. Until now, ACTIS has lacked a practical data processing workflow.…”

“…Third, we showed ACTIS to be applicable to protein−small molecule and protein−aptamer binding pairs. 8,13 Although ACTIS can potentially be used for all molecular complexes, including protein−protein binding pairs, future experimental work needs to be done to show such applicability. Until now, ACTIS has lacked a practical data processing workflow.…”

Introducing Quantitative Assessment of Accuracy for the Equilibrium Constant of Affinity Complexes

“…BSA−fluorescein equilibrium mixtures (EMs) were prepared with the strategy of "new sample-preparation workflow" that was introduced in our previous work. 2 In four ACTIS experiments, the fluorescein concentrations (L0) were fixed to be 0.07, 7.0, 70 and 210 µM, respectively (after correction based on the purity of fluorescein). For the ACTIS experiment with L0 = 0.07 µM, the total BSA concentrations (T0) in the EMs varied from 0 (lowest non-zero T0 = 1.0 µM) to 1000 µM with a total of 10 EMs with different T0 values.…”

Capillary Electrophoresis: A Three-Year Literature Review

“…126,127 With the aid of computer simulation, Wang et al introduced a strategy named "Accurate Constant via Transient Incomplete Separation", which can serve as a reference method for characterizing the affinity of protein− aptamer binding in solution (Figure 3). 128 Strategies were also made for the determination of binding constants in strong complexation. 129,130 The authors demonstrated that the peak shape can be asymmetrical due to electromigration dispersion.…”