Origin and prediction of free-solution interaction studies performed label-free

Abstract

Interaction/reaction assays have led to significant scientific discoveries in the biochemical, medical, and chemical disciplines. Several fundamental driving forces form the basis of intermolecular and intramolecular interactions in chemical and biochemical systems (London dispersion, hydrogen bonding, hydrophobic, and electrostatic), and in the past three decades the sophistication and power of techniques to interrogate these processes has developed at an unprecedented rate. In particular, label-free methods have flourished, such as NMR, mass spectrometry (MS), surface plasmon resonance (SPR), biolayer interferometry (BLI), and backscattering interferometry (BSI), which can facilitate assays without altering the participating components. The shortcoming of most refractive index (RI)-based label-free methods such as BLI and SPR is the requirement to tether one of the interaction entities to a sensor surface. This is not the case for BSI. Here, our hypothesis is that the signal origin for freesolution, label-free determinations can be attributed to conformation and hydration-induced changes in the solution RI. We propose a model for the free-solution response function (FreeSRF) and show that, when quality bound and unbound structural data are available, FreeSRF correlates well with the experiment (R 2 > 0.99, Spearman rank correlation coefficients >0.9) and the model is predictive within ∼15% of the experimental binding signal. It is also demonstrated that a simple mass-weighted dη/dC response function is the incorrect equation to determine that the change in RI is produced by binding or folding event in free solution.backscattering interferometry | assay methodology | molecular interactions | conformation change | hydration change C ontemporary assays enabling single-molecule detection (1,2) have accelerated the sequencing of the human genome (3) and facilitated imaging with extraordinary resolution without labels (4). To most closely approximate the natural state, an interaction assay methodology would interrogate the processes (reaction, molecular interaction, protein folding event, etc.) without perturbation. Label-free chemical and biochemical investigations (5, 6) transduce the desired signal without an exogenous label (fluorescent, radioactive, or otherwise) representing an essential step toward this goal. Many label-free methods require one of the interacting species to be either tethered or immobilized to the sensor surface, introducing a potential perturbation to the natural state of the species (7,8). However, back-scattering interferometry (BSI) is a free-solution label-free technique with the added benefit of sensitivity that rivals fluorescence (9). There are other techniques performed in free solution, such as MS (10, 11) and NMR (12,13) and the widely used isothermal titration calorimetry (ITC) (14, 15). As with NMR, ITC has many advantages, but exhibits modest sensitivity and often requires large sample quantities. Another increasingly popular free-solution approach is microscale thermophoresis (...

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

“…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.…”

Modeling of surface plasmon resonance nanobiosensor with the transverse resonance method

“…The medium is water. The effective permittivity of the solution ε eff based on Maxwell‐Garnett effective medium theory can be written as: …”

Detection Limit for Optically Sensing Specific Protein Interactions in Free-solution

“…In the first approach, values of radius of gyration (R g ) were obtained from the supporting information provided with 6 . The volumes were approximated to using the relation = M w (Da).…”