Reply to Varma: Elucidation of the signal origin for label-free, free-solution interactions

Abstract

In response to the letter submitted by Varma (1), we respectfully disagree with the assertion that our proposed free-solution response function (FreeSRF) model (2) does not explain the signal origin for free-solution measurements. His theoretical considerations are appreciated; however, as we have shown (2), they do not apply here.i) Our empirical model is informative, experimentally consistent, and a reasonable starting point for further elucidating interferometric free-solution, label-free assays. Our approach exhibits a strong correlation (Spearman's rank) between signal magnitude with binding-induced conformation and hydration changes. This correlation held true for a diverse set of binding pairs, ranging widely in affinity and molecular mass. FreeSRF is supported by the published modeling literature that substantiates the use of radius of gyration, R gyr , and solvent-accessible surface area (SASA) as physical parameters to represent intrinsic solution property changes for protein-ligand binding (3, 4). Marsh and Teichmann (4) validate our use of avgSASA in χ, showing it provides an excellent estimation of structural change upon binding. Because SASA also correlates with the amplitude of conformational change, it is reasonable to ascribe the refractive index (RI) signal to this property. We do admit that the dependence on structural and dynamical parameters may be more complex than our linear fit model, so an expanded model may emerge for predicting RI changes in free-solution measurements. ii) Having a large constant term is not reason to discount the validity of an expression, particularly if the physical parameters used in the equation vary widely with respect to the intrinsic property being measured. Although suitable currently, we do anticipate the emergence of a better definition for "E" (the error or disturbance term). iii) Varma's approach, using the Maxwell-Garnett formulation (1), and that used by others (5, 6) predicted ΔRI signal for protein-ligand binding to be below the detection limit (DL) for BSI. This observation is not disputed (2), because we also predicted undetectable binding signals by BSI (<5 × 10 −7 ) using a widely accepted relationship (7) for refractive index increment (RII) calculations (equations S13-S17 and figures S2 and S3 of ref.2). However, quantification of the actual ΔRI signal produced for a binding pair ( (8) and (c) the effect of the medium on the dielectric constant of the protein (and vice versa) (9) is ignored.We conclude that the currently accepted model (5-7) does not accurately predict the signal for freesolution RI measurements. FreeSRF considers hydration and conformation changes and is predictive of both magnitude and directionality for the relative RI change. We hope the "difficulties in replicating BSI experiments" (5, 6) can be reconciled, allowing its successful use, as others have independently shown (10, 11).

Influence of Aptamer Surface Coverage on Small Target Recognition: A SPR and QCM-D Comparative Study

“…This RII deviation, expressed with a correction factor x of the aptamer RII, could be triggered by the conformation transition of the aptamer upon recognition to the l -Tym. Such RII deviations were also reported from backscattering interferometry measurement, for recognition systems undergoing significant conformation or hydration changes . As demonstrated by QCM-D and QCM-D-SE experiments, upon l -Tym recognition, the aptamer sensing layer undergoes both a reduction of its thickness and decrease of its hydration ratio. , …”

“…In addition to the reduction of the sensing layer thickness, the model highlighted the nonadditivity of refractive index increment (RII) for this recognition system: the RII value of the recognition complex is not equal to the sum of the RII values of the two initial partners . This nonadditivity of the RII for the complex was already measured by Bornhop et al in solution but never revealed on the surface. The recognition was also monitored using a quartz crystal microbalance with energy dissipation (QCM-D), and similar affinity constants were measured around 100 μM for both techniques.…”

“…Such RII deviations were also reported from backscattering interferometry measurement, for recognition systems undergoing significant conformation or hydration changes. 11 As demonstrated by QCM-D and QCM-D-SE experiments, upon L-Tym recognition, the aptamer sensing layer undergoes both a reduction of its thickness and decrease of its hydration ratio. 8,9 While the RII value of the aptamer was 0.176 cm 3 •g −1 before the recognition, we have demonstrated that this parameter was increased by 1.48% during the recognition with L-Tym.…”

Real-Time Interferometric Refractive Index Change Measurement for the Direct Detection of Enzymatic Reactions and the Determination of Enzyme Kinetics

“…Studies have shown that BSI can detect low concentrations of both ions and saccharides in capillary electrophoresis [12,13,14] and an early study even used a polariometric-BSI configuration to detect an enzymatic reaction with hydroxybutyrate dehydrogenase [15]. There have also been multiple studies on the molecular interactions between protein-sized molecules using BSI [16], but the effect of such interactions on the bulk refractive index and origin of the observed signal has been debated [12,17,18,19].…”