Amanda Kussrow

Free-Solution, Label-Free Molecular Interactions Studied by Back-Scattering Interferometry

Free-solution, label-free molecular interactions were investigated with back-scattering interferometry in a simple optical train composed of a helium-neon laser, a microfluidic channel, and a position sensor. Molecular binding interactions between proteins, ions and protein, and small molecules and protein, were determined with high dynamic range dissociation constants (Kd spanning six decades) and unmatched sensitivity (picomolar Kds and detection limits of 10,000s of molecules). With this technique, equilibrium dissociation constants were quantified for protein A and immunoglobulin G, interleukin-2 with its monoclonal antibody, and calmodulin with calcium ion Ca2+, a small molecule inhibitor, the protein calcineurin, and the M13 peptide. The high sensitivity of back-scattering interferometry and small volumes of microfluidics allowed the entire calmodulin assay to be performed with 200 picomoles of solute.

Label-free quantification of membrane-ligand interactions using backscattering interferometry

Although membrane proteins are ubiquitous within all living organisms and represent the majority of drug targets, a general method for direct, label-free measurement of ligand binding to native membranes has not been reported. Here we show that backscattering interferometry (BSI) can accurately quantify ligand-receptor binding affinities in a variety of membrane environments. By detecting minute changes in the refractive index of a solution, BSI allows binding interactions of proteins with their ligands to be measured at picomolar concentrations. Equilibrium binding constants in the micromolar to picomolar range were obtained for small- and large-molecule interactions in both synthetic and cell-derived membranes without the use of labels or supporting substrates. The simple and low-cost hardware, high sensitivity and label-free nature of BSI should make it readily applicable to the study of many membrane-associated proteins of biochemical and pharmacological interest.

Measurement of Monovalent and Polyvalent Carbohydrate―Lectin Binding by Back-Scattering Interferometry

Carbohydrate-protein binding is important to many areas of biochemistry. Here, backscattering interferometry (BSI) has been shown to be a convenient and sensitive method for obtaining quantitative information about the strengths and selectivities of such interactions. The surfaces of glass microfluidic channels were covalently modified with extravidin, to which biotinylated lectins were subsequently attached by incubation and washing. The binding of unmodified carbohydrates to the resulting avidin-immobilized lectins was monitored by BSI. Dose-response curves that were generated within several minutes and were highly reproducible in multiple wash/measure cycles provided adsorption coefficients that showed mannose to bind to concanavalin A (conA) with 3.7 times greater affinity than glucose consistent with literature values. Galactose was observed to bind selectively and with similar affinity to the lectin BS-1. The avidities of polyvalent sugar-coated virus particles for immobilized conA were much higher than monovalent glycans, with increases of 60-200 fold per glycan when arrayed on the exterior surface of cowpea mosaic virus or bacteriophage Qbeta. Sugar-functionalized PAMAM dendrimers showed size-dependent adsorption, which was consistent with the expected density of lectins on the surface. The sensitivity of BSI matches or exceeds that of surface plasmon resonance and quartz crystal microbalance techniques, and is sensitive to the number of binding events, rather than changes in mass. The operational simplicity and generality of BSI, along with the near-native conditions under which the target binding proteins are immobilized, make BSI an attractive method for the quantitative characterization of the binding functions of lectins and other proteins.

Interferometric methods for label-free molecular interaction studies.

Interferometry has long been a valuable tool for measuring both the micro- and macroworlds, from solar dimensions to DNA interactions. On the biosensing frontier, a number of interferometric systems have been developed which offer significant advantages over traditional methods for measuring molecular interactions. In particular, interferometry enables the detection and study of binding events without the use of expensive and disruptive labels. Recent advancements in technologies utilizing interferometry have made significant improvements in the last several years. For example, diffraction optics has been improved through the use of nanowire gratings and multiple geometries, resulting in enhanced multiplexing and an increased instrument dynamic range. Innovations in dual polarization have demonstrated the ability to measure kinetics while simultaneously capturing structural information about the binding partners. Waveguiding microresonators, such as the microring resonator, have shown exquisite sensitivities while maintaining a straightforward and inexpensive multiplex format. New biosensing technologies have also come forward, such as backscattering interferometry which uniquely offers the additional advantage of measuring binding interactions in free solution while still using small amounts of sample.

Evolution and protein packaging of small-molecule RNA aptamers.

A high-affinity RNA aptamer (K(d) = 50 nM) was efficiently identified by SELEX against a heteroaryldihydropyrimidine structure, chosen as a representative drug-like molecule with no cross reactivity with mammalian or bacterial cells. This aptamer, its weaker-binding variants, and a known aptamer against theophylline were each embedded in a longer RNA sequence that was encapsidated inside a virus-like particle by a convenient expression technique. These nucleoprotein particles were shown by backscattering interferometry to bind to the small-molecule ligands with affinities similar to those of the free (nonencapsidated) aptamers. The system therefore comprises a general approach to the production and sequestration of functional RNA molecules, characterized by a convenient label-free analytical technique.

Baclofen and Other GABAB Receptor Agents Are Allosteric Modulators of the CXCL12 Chemokine Receptor CXCR4

CXCR4, a receptor for the chemokine CXCL12 (stromal-cell derived factor-1α), is a G-protein-coupled receptor (GPCR), expressed in the immune and CNS and integrally involved in various neurological disorders. The GABAB receptor is also a GPCR that mediates metabotropic action of the inhibitory neurotransmitter GABA and is located on neurons and immune cells as well. Using diverse approaches, we report novel interaction between GABAB receptor agents and CXCR4 and demonstrate allosteric binding of these agents to CXCR4. First, both GABAB antagonists and agonists block CXCL12-elicited chemotaxis in human breast cancer cells. Second, a GABAB antagonist blocks the potentiation by CXCL12 of high-threshold Ca2+ channels in rat neurons. Third, electrophysiology in Xenopus oocytes and human embryonic kidney cell line 293 cells in which we coexpressed rat CXCR4 and the G-protein inward rectifier K+ (GIRK) channel showed that GABAB antagonist and agonist modified CXCL12-evoked activation of GIRK channels. To investigate whether GABAB ligands bind to CXCR4, we expressed this receptor in heterologous systems lacking GABAB receptors and performed competition binding experiments. Our fluorescent resonance energy transfer experiments suggest that GABAB ligands do not bind CXCR4 at the CXCL12 binding pocket suggesting allosteric modulation, in accordance with our electrophysiology experiments. Finally, using backscattering interferometry and lipoparticles containing only the CXCR4 receptor, we quantified the binding affinity for the GABAB ligands, confirming a direct interaction with the CXCR4 receptor. The effect of GABAergic agents on CXCR4 suggests new therapeutic potentials for neurological and immune diseases.

Comparison of Free-Solution and Surface-Immobilized Molecular Interactions Using a Single Platform

While it is generally accepted that surface immobilization affects the binding properties of proteins, it has been difficult to quantify these effects due to the lack of technology capable of making affinity measurements with species tethered and in free solution on a single platform. Further, quantifying the interaction of binding pairs with widely differing masses has also been challenging, particularly when it is desirable to tether the high molecular weight protein. Here we describe the use of backscattering interferometry (BSI) to quantify the binding affinity of mannose and glucose to concanavalin A (ConA), a 106 KDa homotetramer protein, in free solution using picomoles of the protein. Using the same platform, BSI, we then studied the effect on the binding constants of the ConA-carbohydrate interactions upon chemically immobilizing ConA on the sensor surface. By varying the distances (0, 7.17, and 20.35 nm) of the ConA tether and comparing these results to the free-solution measurements, it has been possible to quantify the effect that protein immobilization has on binding. Our results indicate that the apparent binding affinity of the sugar-lectin pair increases as the distance between ConA and the surface decreases. These observations could lend insight as to why the affinity values reported in the literature sometimes vary significantly from one measurement technique to another.

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

Significance Chemical and biomedical sciences depend heavily on interaction assays, particularly those providing structural insights. Here, we show interferometric, free-solution, label-free studies report conformation and hydration changes, and present a new way for interpreting these methods. Intrinsic property changes are the mechanism allowing for unprecedented sensitivities (picomolar to femtomolar) in complex milieu, even when individual binding partners are undetectable. We establish that the existing theory for label-free assay methods such as surface plasmon resonance (SPR) is not applicable and propose a model for the free-solution response function (FreeSRF), validated and highly predictive when combined with quality structural data and reliable calculations of solvent-addressable surface area. The model allows for interpretation of solution-phase, label-free interactions and could facilitate obtaining structural information from a simple mix-and-read assay. 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 free-solution, 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 (R2 > 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.

The potential of backscattering interferometry as an in vitro clinical diagnostic tool for the serological diagnosis of infectious disease

Backscattering interferometry enables the detection of syphilis antibody-antigen interactions in the presence of human serum, showing promise as a diagnostic tool for the serological diagnosis of infectious disease with potentially quantitative capabilities.

Free-solution interaction assay of carbonic anhydrase to its inhibitors using back-scattering interferometry

Back‐scattering interferometry (BSI) is a label‐free, free‐solution, small‐volume technique used for characterizing binding interactions, which is also relevant to a growing number of biosensing applications including drug discovery. Here, we use BSI to characterize the interaction of carbonic anhydrase enzyme II with five well‐known carbonic anhydrase enzyme II inhibitors (±sulpiride, sulfanilamide, benzene sulfonamide, dansylamide, and acetazolamide) in the presence of DMSO. Dissociation constants calculated for each interaction were consistent with literature values previously obtained using surface plasmon resonance and fluorescence‐based competition assays. Results demonstrate the potential of BSI as a drug‐screening tool which is fully compatible with DMSO and does not require immobilization or labeling, therefore allowing binding interactions to be characterized in the native state. BSI has the potential for reducing labor costs, sample consumption, and assay time while providing enhanced reliability over existing techniques.