Measuring Aptamer Equilibria Using Gradient Micro Free Flow Electrophoresis

Abstract

Gradient micro free flow electrophoresis (μFFE) was used to observe the equilibria of DNA aptamers with their targets (IgE or HIVRT) across a range of ligand concentrations. A continuous stream of aptamer was mixed online with an increasing concentration of target and introduced into the μFFE device, which separated ligand-aptamer complexes from the unbound aptamer. The continuous nature of μFFE allowed the equilibrium distribution of aptamer and complex to be measured at 300 discrete target concentrations within 5 minutes. This is a significant improvement in speed and precision over affinity capillary electrophoresis (ACE) assays. The dissociation constant of the aptamer-IgE complex was estimated to be 48± 3 nM. The high coverage across the range of ligand concentrations allowed complex stoichiometries of the aptamer-HIVRT complexes to be observed. Nearly continuous observation of the equilibrium distribution from 0 to 500 nM HIVRT revealed the presence of complexes with 3:1 (aptamer:HIVRT), 2:1 and 1:1 stoichiometries.

Methods for measuring aptamer-protein equilibria: A review

“…Microchip CE devices with flow gated injection shortens separations time to as little as 10 seconds, simplifying measurements of aptamers with faster dissociation rates [128, 129]. Micro free flow electrophoresis (μFFE) can be used to monitor the bound and free aptamer across a wide protein concentration range using gradient pumping, generating over 300 data points across the binding curve in as little as five minutes [89]. PDMS microfluidic chips with multiple channels and SPR imaging are able to test multiple aptamers’ affinity toward one target at the same time [130].…”

“…Another possible consequence of binding involves a conformation change and formation of a donor-acceptor complex at the ground state, resulting in a shift in the fluorescent emission profile [85]. As a result, the potential for either fluorescence enhancement or quenching is possible depending on the specific system under study [86–89]. By subtracting fluorescence signals from free ligand and background one can obtain intensity changes and calculate binding fractions.…”

Reassemblable quasi‐chip free‐flow electrophoresis with simple heating dispersion for rapid micropreparation of trypsin in crude porcine pancreatin

“…In 1994, Raymond et al [23] developed the first C-FFE device for bioseparation. In 2006-2010, Bowser and coworkers conducted a series of fundamental studies on the flow controlling [24], band and resolution optimizations [25], mitochondria mobility determination [26], and aptamer equilibria measurement [27]. A lot of works have demonstrated that C-FFE could be excellently used for the analyses of mitochondria [26], aptamers [27], and proteins [28] as well as the estimation of equilibrium constants [29].…”

Advances in steady-state continuous-flow purification by small-scale free-flow electrophoresis

“…However, due to short-term steady-state conditions and μFFE dimensions, separation is limited to small sample volumes and the sample components are restricted in size to avoid clogging. As a result, rather than being used exclusively as a preparative technique, µFFE found an important purpose as an analytical tool to measure equilibrium constants of biomolecular interactions [88]. µFFE was also used to separate and to select DNA aptamers for a target molecule [89].…”