Current practical methods for finding the equilibrium dissociation constant, K d ,o fp rotein-small molecule complexes have inherent sources of inaccuracy.I ntroduced here is "accurate constant via transient incomplete separation" (ACTIS), which appears to be free of inherent sources of inaccuracy.C onceptually,as hort plug of the pre-equilibrated protein-small molecule mixture is pressure-propagated in ac apillary,c ausing fast transient incomplete separation of the complex from the unbound small molecule.Asuperposition of signals from these two components is measured near the capillary exit and used to calculate af raction of unbound small molecule,which,inturn, is used to calculate K d . Herein the validity of ACTIS is proven theoretically,i ts accuracy is verified by computer simulation, and its practical use is demonstrated. ACTIS has the potential to become ar eference-standardm ethod for determining K d values of protein-small molecule complexes.Reversible binding of proteins (P) to small-molecule ligands (L) plays an important role in the regulation of cellular processes. [1] In addition, most therapeutic targets are proteins, [2] and drugs are developed to form stable PL complexes with them:Complex stability is characterized by the equilibrium dissociation constant K d ,which is defined as:

Analytical methods may not have reference standards required for testing their accuracy. We postulate that accuracy of an analytical method can be assessed in the absence of reference standards in silico if the method is built upon deterministic processes. A deterministic process can be precisely computer-simulated thus allowing virtual experiments with virtual reference standards. Here, we apply this in silico approach to study "Accurate Constant via Transient Incomplete Separation" (ACTIS), a method for finding the equilibrium dissociation constant (K d) of protein-small molecule complexes. ACTIS is based on a deterministic process: molecular diffusion of the interacting protein-small molecule pair in a laminar pipe flow. We used COMSOL software to construct a virtual ACTIS setup with a fluidic system mimicking that of a physical ACTIS instrument. Virtual ACTIS experiments performed with virtual samples-mixtures of a protein and a small molecule with defined rate constants and, thus, K d of their interaction-allowed us to assess ACTIS accuracy by comparing the determined K d value to the input K d value. Further, the influence of multiple system parameters on ACTIS accuracy was investigated. Within multi-fold ranges of parameters, the values of K d did not deviate from the input K d values by more than a factor of 1.25 strongly suggesting that ACTIS is intrinsically accurate and that its accuracy is robust. Accordingly, further development of ACTIS can focus on achieving high reproducibility and precision. We foresee that in silico accuracy assessment, demonstrated here with ACTIS, will be applicable to other analytical methods built upon deterministic processes. File list (5) download file view on ChemRxiv manuscript.pdf (741.60 KiB) download file view on ChemRxiv supportinginformation.pdf (2.21 MiB) download file view on ChemRxiv thumbnail.png (161.31 KiB) download file view on ChemRxiv models.zip (1.29 MiB)

Current practical methods for finding the equilibrium dissociation constant, K d ,o fp rotein-small molecule complexes have inherent sources of inaccuracy.I ntroduced here is "accurate constant via transient incomplete separation" (ACTIS), which appears to be free of inherent sources of inaccuracy.C onceptually,as hort plug of the pre-equilibrated protein-small molecule mixture is pressure-propagated in ac apillary,c ausing fast transient incomplete separation of the complex from the unbound small molecule.Asuperposition of signals from these two components is measured near the capillary exit and used to calculate af raction of unbound small molecule,which,inturn, is used to calculate K d . Herein the validity of ACTIS is proven theoretically,i ts accuracy is verified by computer simulation, and its practical use is demonstrated. ACTIS has the potential to become ar eference-standardm ethod for determining K d values of protein-small molecule complexes.Reversible binding of proteins (P) to small-molecule ligands (L) plays an important role in the regulation of cellular processes. [1] In addition, most therapeutic targets are proteins, [2] and drugs are developed to form stable PL complexes with them:Complex stability is characterized by the equilibrium dissociation constant K d ,which is defined as:

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.

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.

The equilibrium dissociation constant (Kd) characterizes stability of non-covalent molecular complexes. Determining Kd for highly stable complexes may be extremely inaccurate if the ratio between the concentration of the limiting component (L0) and the a priori unknown value of Kd exceeds an unknown threshold value (aka threshold ratio). The only known approach to reveal this kind of inaccuracy in Kd requires building multiple experimental binding isotherms; it is resource intensive and, therefore, used very rarely. Here we introduce a single-isotherm approach for assessing Kd accuracy via determining the value of L0/Kd, estimating the threshold ratio, and comparing L0/Kd to the threshold ratio. In this proof-of-concept work, we present the theoretical basis and develop a step-by-step algorithm for our single-isotherm approach. We also demonstrate the experimental use of the developed algorithm.

The determination of accurate equilibrium dissociation constants, K d, of protein–small molecule complexes is important but challenging as all established methods have inherent sources of inaccuracy. Accurate Constant via Transient Incomplete Separation (ACTIS) is a new method for K d determination using transient incomplete separation of the complex from the unbound small molecule in a pressure-driven flow inside a capillary. ACTIS is accurate, and its accuracy is invariant to variations in geometries of both the fluidic system and the flow. Furthermore, ACTIS is implemented using a simple fluidic system supporting its accuracy and providing a simple-to-follow/copy template for instrumentation. Despite the simple and robust instrumentation/acquisition, the current data processing workflow is cumbersome, time consuming, and prone to hard-to-trace human errors therefore hindering ACTIS’ ability to become a practical reference method for K d determination. This technical note describes a streamlined workflow for processing ACTIS data; the workflow is implemented as a set of open-source software tools called prACTISed (). These tools allow all steps of data processing to be performed in a fast and straightforward fashion. These practical software tools complement the simple instrumentation serving both developers and users of ACTIS.

The determination of accurate equilibrium dissociation constants Kd of protein–small molecule complexes is important but challenging as all established methods have inherent sources of inaccuracy. ACTIS is a new method for Kd determination using transient incomplete separation of the complex from the unbound small molecule in a pressure-driven flow inside a capillary. ACTIS is accurate and its accuracy is invariant to variations in geometries of both the fluidic system and the flow. Furthermore, ACTIS is implemented using a simple fluidic system supporting its accuracy and providing a simple-to-follow/copy template for instrumentation. Despite the simple and robust instrumentation/acquisition, the current data processing workflow is cumbersome, time-consuming, and prone to hard-to-trace human errors therefore hindering ACTIS’ ability to become a practical reference method for Kd determination. This technical note describes a streamlined workflow for processing ACTIS data; the workflow is implemented as a set of open-source software tools called prACTISed (https://github.com/prACTISedProgram/prACTISed). These tools allow all steps of data processing to be performed in fast and straightforward fashion. These practical software tools complement the simple instrumentation serving both developers and users of ACTIS.