The NEOtrap – en route with a new single-molecule technique

Schmid, Sonja; Dekker, Cees

doi:10.1016/j.isci.2021.103007

Cited by 11 publications

(9 citation statements)

References 99 publications

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“…We next wanted to test O(GGS) 4 and O(PA) 6 using our amalgamated ND-BLI approach for further validation. We anticipated that the ND-BLI results for these 12-residue tethercontaining sensors would yield weaker interactions than O(GGS) 5 and O(PA) 8 because they have an increased tether restraint.…”

Section: Resultsmentioning

confidence: 99%

“…A single nanopore is a versatile sensing element for numerous tasks in protein analytics. − Significant progress has been accomplished in basic research and biosensing technology using nanopores − fabricated in various scaffolds and materials. − The readout signal in these sensors is the transmembrane current through a nanopore . Key advantages that make this approach influential include the following: (i) this label-free method probes time-resolved molecular events at a single-molecule level; − (ii) the nanopore structure and composition can be altered with atomic precision; , (iii) nanopores are amenable to automated microelectrode recording technologies; − (iv) electrical recordings with single nanopores can be conducted in a broad dynamic range of interactions and analyte concentrations; (v) specific and sensitive detection can be performed in challenging heterogeneous solutions, such as biofluids, − or in complex mixtures of proteins .…”

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confidence: 99%

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Evaluation of Nanopore Sensor Design Using Electrical and Optical Analyses

et al. 2023

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Nanopores are currently utilized as powerful tools for single-molecule protein sensing. The reporting signal typically requires protein analytes to enter the nanopore interior, yet a class of these sensors has emerged that allows targeted detection free in solution. This tactic eliminates the spatial limitation of nanopore confinement. However, probing proteins outside the nanopore implies numerous challenges associated with transducing the physical interactions in the aqueous phase into a reliable electrical signature. Hence, it necessitates extensive engineering and tedious optimization routes. These obstacles have prevented the widespread adoption of these sensors. Here, we provide an experimental strategy by developing and validating single-polypeptide-chain nanopores amenable to single-molecule and bulk-phase protein detection approaches. We utilize protein engineering, as well as nanopore and nanodisc technologies, to create nanopore sensors that can be integrated with an optical platform in addition to traditional electrical recordings. Using the optical modality over an ensemble of detectors accelerates these sensors’ optimization process for a specific task. It also provides insights into how the construction of these single-molecule nanopore sensors influences their performance. These outcomes form a basis for evaluating engineered nanopores beyond the fundamental limits of the resistive-pulse technique.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Evaluation of Nanopore Sensor Design Using Electrical and Optical Analyses

et al. 2023

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“…However, the underlying kinetic rates are different (Schmid and Hugel 2020 ), demonstrating the added functional insight gained from single-molecule kinetics data. In addition, she harnesses new label-free nanopore techniques (Schmid and Dekker 2021a ) to study Hsp90, for example using their recently developed NEOtrap (Schmid et al 2021 ), to tackle persistent questions relating to the Hsp90 mechanism with new time-resolved single-molecule techniques (Schmid and Dekker 2021b ).…”

Section: Hsp90 Structure–function Relationshipsmentioning

confidence: 99%

Tenth International Symposium on the Hsp90 chaperone machine

Edkins

Zweckstetter

Sawarkar

2023

Cell Stress and Chaperones

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Hsp90 is a molecular chaperone responsible for regulating proteostasis under physiological and pathological conditions. Its central role in a range of diseases and potential as a drug target has focused efforts to understand its mechanisms and biological functions and to identify modulators that may form the basis for therapies. The 10th international conference on the Hsp90 chaperone machine was held in Switzerland in October 2022. The meeting was organized by Didier Picard (Geneva, Switzerland) and Johannes Buchner (Garching, Germany) with an advisory committee of Olivier Genest, Mehdi Mollapour, Ritwick Sawarkar, and Patricija van Oosten-Hawle. This was a much anticipated first in-person meeting of the Hsp90 community since 2018 after the COVID-19 pandemic led to the postponement of the 2020 meeting. The conference remained true to the tradition of sharing novel data ahead of publication, providing unparalleled depth of insight for both experts and newcomers to the field.

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“…Few techniques have the ability to sense single biomolecules in a label-free manner, and even fewer can do so in solution and at room temperature. The recently developed nanopore electro-osmotic trap (NEOtrap) is such a label-free single-molecule technique that can trap and study proteins one by one. , As shown in Figure a, the NEOtrap consists of a DNA-origami sphere that is docked onto a passivated solid-state nanopore when a positive bias voltage is applied (to the trans side). Once docked, the highly negatively charged DNA-origami sphere generates an electro-osmotic flow (EOF) by which a target protein can be trapped (Figure b).…”

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confidence: 99%

Orientation-Locked DNA Origami for Stable Trapping of Small Proteins in the Nanopore Electro-Osmotic Trap

et al. 2022

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Nanopores are versatile single-molecule sensors offering a simple label-free readout with great sensitivity. We recently introduced the nanopore electro-osmotic trap (NEOtrap) which can trap and sense single unmodified proteins for long times. The trapping is achieved by the electro-osmotic flow (EOF) generated from a DNA-origami sphere docked onto the pore, but thermal fluctuations of the origami limited the trapping of small proteins. Here, we use site-specific cholesterol functionalization of the origami sphere to firmly link it to the lipid-coated nanopore. We can lock the origami in either a vertical or horizontal orientation which strongly modulates the EOF. The optimized EOF greatly enhances the trapping capacity, yielding reduced noise, reduced measurement heterogeneity, an increased capture rate, and 100-fold extended observation times. We demonstrate the trapping of a variety of single proteins, including small ones down to 14 kDa. The cholesterol functionalization significantly expands the application range of the NEOtrap technology.

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The NEOtrap – en route with a new single-molecule technique

Cited by 11 publications

References 99 publications

Evaluation of Nanopore Sensor Design Using Electrical and Optical Analyses

Evaluation of Nanopore Sensor Design Using Electrical and Optical Analyses

Tenth International Symposium on the Hsp90 chaperone machine

Orientation-Locked DNA Origami for Stable Trapping of Small Proteins in the Nanopore Electro-Osmotic Trap

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