Interferometric Methods for Label-Free Molecular Interaction Studies

Kussrow, Amanda; Enders, Carolyn S.; Bornhop, Darryl J.

doi:10.1021/ac202812h

Cited by 62 publications

(63 citation statements)

References 133 publications

(365 reference statements)

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“…Detection limits vary for SPR, but consistently reach ΔRI = 10 −6 . In our hands, the BSI detection limit is ΔRI = 10 −6 or 10-fold below this level (19,20,62). Therefore, using proper methodology the signal to noise ratio (S/N) of our interferometer should enable molecular interactions to be measured.…”

Section: Conformation and Hydration Changes Are The Origin Of Free-somentioning

confidence: 95%

“…The importance of these preliminary studies was not more fully realized until 2007, when our group showed that binding events, such as ion-protein, protein-protein, and small molecule-protein interactions, could be measured using a RI technique in free solution and without labels (9). Numerous examples have validated that free-solution measurements by interferometry can be used to quantity interactions of widely different affinities (micromolar to picomolar) and on interacting pairs with significant mass differences (>10,000-fold) (18)(19)(20). Although we, and others, have postulated the origin of the free-solution signal, no explicit explanation for the physical phenomenon has emerged.…”

Section: Introductionmentioning

confidence: 99%

“…However, for MST to operate label-free, one of the binding partners must have a significant absorption/fluorescence cross-section (16,17). BSI represents an attractive alternative to these methods because of its high sensitivity, small sample volume requirement, optical simplicity, and broad applicability (18)(19)(20)(21).…”

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confidence: 99%

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

Bornhop

Kammer

Kussrow

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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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 (...

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Section: Conformation and Hydration Changes Are The Origin Of Free-somentioning

confidence: 95%

Section: Introductionmentioning

confidence: 99%

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

Bornhop

Kammer

Kussrow

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Back-scattering interferometry (BSI) is an emerging technique for measuring molecular interactions in solution without the need for labeling or immobilization [80]. The platform consists of a collimated coherent light source (HeNe laser), a capillary or microfluidic chip containing the sample, and a CCD array detector.…”

Section: Back-scattering Interferometrymentioning

confidence: 99%

Protein microarrays, biosensors, and cell-based methods for secretome-wide extracellular protein–protein interaction mapping

Gonzalez¹

2012

Methods

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Approximately one quarter of all human genes encode proteins that function in the extracellular space or serve to bridge the extracellular and intracellular environments. Physical associations between these secretome proteins serve to regulate a wide range of biological activities and consequently represent important therapeutic targets. Moreover, some extracellular proteins are targeted by pathogens to allow host access or immune evasion. Despite the importance of extracellular protein-protein interactions, our knowledge in this area has remained sparse. Weak affinities and low abundance have often hindered efforts to identify these interactions using traditional methods such as biochemical purification and cDNA library expression cloning. Moreover, current large-scale protein-protein interaction mapping techniques largely under represent extracellular protein-protein interactions. This review highlights emerging biosensor and protein microarray technology, along with more traditional cell-based techniques, that are compatible with secretome-wide screens for extracellular protein-protein interaction discovery. A combination of these approaches will serve to rapidly expand our knowledge of the extracellular protein-protein interactome.

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“…En una revisión más reciente [Kussrow, 2012] se analizan en particular los métodos interferométricos aplicados a la implementación de sistemas biosensores. Esta revisión, uno de cuyos autores es el autor de los primeros estudios de backscattering en capilares y canales rectangulares, es particularmente interesante por la revisión de los últimos logros de esta técnica en cuanto a sensibilidad y límite de detección.…”

Section: La Celdas Biofotónicas Bicellunclassified

Caracterización y optimización de estructuras biofotónicas y técnicas avanzadas de interrogación para desarrollo de biosensores ópticos

Hueros¹

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Interferometric Methods for Label-Free Molecular Interaction Studies

Cited by 62 publications

References 133 publications

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

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

Protein microarrays, biosensors, and cell-based methods for secretome-wide extracellular protein–protein interaction mapping

Caracterización y optimización de estructuras biofotónicas y técnicas avanzadas de interrogación para desarrollo de biosensores ópticos

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