Template Instrumentation for “Accurate Constant via Transient Incomplete Separation”

Abstract

Accurate Constant via Transient Incomplete Separation (ACTIS) is a new method for finding the equilibrium dissociation constant K d of a protein− small molecule complex based on transient incomplete separation of the complex from the unbound small molecule in a capillary. This separation is caused by differential transverse diffusion of the complex and the small molecule in a pressure-driven flow. The advection-diffusion processes underlying ACTIS can be described by a system of partial differential equations allowing for a virtual ACTIS instrument to be built and ACTIS to be studied in silico. The previous in silico studies show that large variations in the fluidic system geometry do not affect the accuracy of K d determination, thus, proving that ACTIS is conceptually accurate. The conceptual accuracy does not preclude, however, instrumental inaccuracy caused by run-to-run signal drifts. Here we report on assembling a physical ACTIS instrument with a fluidic system that mimics the virtual one and proving the absence of signal drifts. Furthermore, we confirmed method ruggedness by assembling a second ACTIS instrument and comparing the results of experiments performed with both instruments in parallel. Despite some unintentional differences between the instruments (caused by tolerances in sizes, positions, etc.) and noticeable differences in their respective separagrams, we found that the K d values determined for identical samples with these instruments were equal. Conclusively, the fluidic system presented here can serve as a template for reliable ACTIS instrumentation.

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

“…Instabilities in a representative physical ACTIS setup have been recently studied in detail. 26 Instrumental low-frequency variations in the flow rate and detector response were found to cause a run-to-run signal variation (RSD) of ∼2%. Relative noise of the detector (high frequency) should be considered separately from these variations as unlike them it depends on the signal value, which, in turn, is inversely proportional to [L] 0 .…”

“…There is a universally accepted view that TIS is only applicable to species greatly different in size. Therefore, all examples of TIS in the literature relate to species, which differ in the diffusion coefficient by a factor of 10 or more. − Such a predisposition made previous ACTIS efforts to focus on protein–small-molecule complexes (diffusion coefficients of a small molecule and a protein–small-molecule complex differ by approximately a factor of 10). The goal of this work was to understand if the TIS of protein–large ligand complex from the unbound ligand could be detected and used for K d determination for such complexes.…”

“…ACTIS Instrumentation. The ACTIS instrumental setup was identical to the previously published one, 26 except for the following modifications (see schematics in Figure S1). First, the capillary for draining waste was replaced by a 7.0 cm-long "sample aspiration capillary".…”

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

“…If separation of L from TL is incomplete, then only a cumulative signal S from them can be measured. If the signals from L and TL do not interfere (which is true in most detection modes), then the cumulative signal follows the principle of superposition: , S = S L [ L ] [ L ] 0 + S TL [ TL ] [ L ] 0 …”

“…where φ is a of signal changes when L binds to T, for example, the quantum yield of TL relative to that of L. If separation of L from TL is incomplete, then only a cumulative signal S from them can be measured. If the signals from L and TL do not interfere (which is true in most detection modes), then the cumulative signal follows the principle of superposition: 22,23…”