Single-molecule protein sensing in a nanopore: a tutorial

Varongchayakul, Nitinun; Song, Jiaxi; Meller, A.; Grinstaff, Mark W.

doi:10.1039/c8cs00106e

Cited by 235 publications

(239 citation statements)

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“…While the volume-based model in Equation 2 accurately describes streptavidin's translocation at low voltage, it does not address the effects of protein orientation (relative to the pore axis) or of conformational changes which become increasingly likely at higher voltages that favor protein shearing. (46,47) Recently, Yusko(19) and Houghtaling et al (40) developed a model linked to Maxwell's derivation:…”

Section: Monovalent Streptavidinmentioning

confidence: 99%

“…(60, 61) Further, the stochastic nature of translocation-induced conformational deformation produces a broader distribution of dwell times. (47) The most likely explanation for the decreased blockage depth and increased dwell time is a partial unfolding or restructuring of the protein under the larger force of a 600 mV applied bias.…”

Section: Monovalent Streptavidinmentioning

confidence: 99%

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Mapping shifts in nanopore signal to changes in protein and protein-DNA conformation

Carlsen

Tabard‐Cossa

2020

Preprint

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Solid-state nanopores have been used extensively in biomolecular studies involving DNA and proteins. However, the interpretation of signals generated by the translocation of proteins or protein-DNA complexes remains challenging. Here, we investigate the behavior of monovalent streptavidin and the complex it forms with short biotinylated DNA over a range of nanopore sizes, salts and voltages. We describe a simple geometric model that is broadly applicable and employ it to explain observed variations in conductance blockage and dwell time with experimental conditions. The general approach developed here underscores the value of nanopore-based protein analysis and represents progress toward the interpretation of complex translocation signals. STATEMENT OF SIGNIFICANCENanopore sensing allows investigation of biomolecular structure in aqueous solution, including electricfield-induced changes in protein conformation. This nanopore-based study probes the tetramer-dimer transition of streptavidin, observing the effects of increasing voltage with varying salt type and concentration. Binding of biotinylated DNA to streptavidin boosts the complex's structural integrity, while complicating signal analysis. We describe a broadly applicable geometric approach that maps stepwise changes in the nanopore signal to real-time conformational transitions. These results represent important progress toward accurate interpretation of nanopore signals generated by macromolecular complexes.

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Section: Monovalent Streptavidinmentioning

confidence: 99%

Section: Monovalent Streptavidinmentioning

confidence: 99%

Mapping shifts in nanopore signal to changes in protein and protein-DNA conformation

Carlsen

Tabard‐Cossa

2020

Preprint

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“…As expected, we observed transient dips in the current through the bare pore upon injection of the proteins, which we attribute to single-molecule translocations of the analyte molecules. As is typical in nanopore experiments, translocation events yield current blockades with a characteristic amplitude and dwell time, where the former relates to the size of the molecule occupying the pore and the latter generally depends on specific interaction between the translocating molecule and the pore wall 55 . Next, we repeated the experiment under identical conditions on the same pore after coating with NupX took place ( Figure 3b).…”

Section: Single-molecule Translocation Experiments With Nupx-coated Nmentioning

confidence: 99%

A designer FG-Nup that reconstitutes the selective transport barrier of the Nuclear Pore Complex

Fragasso

Vries

Sluis

et al. 2020

Preprint

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Nuclear Pore Complexes (NPCs) regulate bidirectional transport between the nucleus and the cytoplasm. Intrinsically disordered FG-Nups line the NPC lumen and form a selective barrier, where transport of most proteins is inhibited whereas specific transporter proteins freely pass. The mechanism underlying selective transport through the NPC is still debated. Here, we reconstitute the selective behaviour of the NPC bottom-up by introducing a rationally designed artificial FG-Nup that mimics natural Nups. Using QCM-D, we measure a strong affinity of the artificial FG-Nup brushes to the transport receptor Kap95, whereas no binding occurs to cytosolic proteins such as BSA. Solid-state nanopores with the artificial FG-Nups lining their inner walls support fast translocation of Kap95 while blocking BSA, thus demonstrating selectivity. Coarse-grained molecular dynamics simulations highlight the formation of a selective meshwork with densities comparable to native NPCs. Our findings show that simple design rules can recapitulate the selective behaviour of native FG-Nups and demonstrate that no specific spacer sequence nor a spatial segregation of different FG-motif types are needed to create functional NPCs. IntroductionNucleocytoplasmic transport is orchestrated by the Nuclear Pore Complex (NPC), which imparts a selective barrier to biomolecules 1,2 . The NPC is a large eightfold-symmetric protein complex (with a size of ~52 MDa in yeast and ~112 MDa in vertebrates) that is embedded within the nuclear envelope and comprises ~30 different types of Nucleoporins ('Nups') 3,4 . Intrinsically disordered proteins, termed FG-Nups, line the central channel of the NPC. FG-Nups are characterized by the presence of phenylalanine-glycine (FG) repeats separated by spacer sequences 5 and they are highly conserved throughout species 6 . FG-Nups carry out a dual function: By forming a dense barrier (100-200 mg/mL) within the NPC lumen, they allow passage of molecules in a size-selective manner 7-10 . Small molecules can freely diffuse through, whereas larger particles are generally excluded 11 . At the same time, FG-Nups mediate the transport of large NTR-bound (Nuclear Transport Receptor) cargoes across the NPC through transient hydrophobic interactions between FG repeats and hydrophobic pockets on the convex side of NTRs 12 . Various models have been developed in order to connect the physical properties of FG-Nups to the size-selective properties of the NPC central channel, e.g. the 'virtual-gate' 13 , 'selective phase' 14,15 , 'reduction of dimensionality' 16 , 'kap-centric' 17-19 , 'polymer brush' 20 , and 'forest' 5 models.As is evident from the multitude of transport models, no consensus on the NPC transport mechanisms has yet been reached.The NPC is highly complex in its architecture and dynamics, being constituted by many different Nups that simultaneously interact with multiple transiting cargoes and NTRs. In fact, translocating cargoes may amount to almost half of the mass of the central channel, so they may be considered an ...

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“…1384pF (1) CSi-B2 +C Si Table S3. The calculation of the sapphire nanopore capacitance in Figure S1d.…”

Section: Csi-b1mentioning

confidence: 99%

“…Further, we have demonstrated that a sapphire nanopore 23 chip (~7 nm pore diameter in a 30 nm thick and 70 µm wide SiN membrane) has more than two-24 order-of-magnitude smaller device capacitance (10 pF) compared to a float-zone Si based 25 nanopore chip (4 nm pore in 23 nm thick and ~4 µm wide SiN membrane, ~1.3 nF), despite having 26 a 100 times larger membrane area. The sapphire chip has a current noise of 18 pA over 100 kHz 27 bandwidth at a 50 mV bias, much smaller than that from the Si chip (46 pA) and only slightly 28 Introduction 34 Solid-state nanopores have attracted a lot of interest as a potentially high-speed, portable and 35 low-cost solution for detecting a variety of biomolecules, such as proteins 1,2,3,4 , RNA 5, 6, 7 and 36 DNA 8, 9, 10 , and studying molecular interactions 11,12 . However, fundamental limitations in design 37 and manufacturing of low-noise nanopore devices still remain.…”

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confidence: 99%

Sapphire-Supported Nanopores for Low-Noise DNA Sensing

Xia

Zuo

Paudel

et al. 2020

Preprint

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12Solid-state nanopores have broad applications in single-molecule biosensing and diagnostics, but 13 their high electrical noise associated with a large device capacitance has seriously limited both 14 their sensing accuracy and recording speed. Current strategies to mitigate the noise has focused on 15introducing insulating materials (such as polymer or glass) to decrease the device capacitance, but 16 the complex process integration schemes diminish the potential to reproducibly create such 17larger than the open-headstage system noise (~11 pA). Further, we demonstrate that the sapphire 29 nanopore chip outperforms the Si chip with a higher signal-to-noise ratio (SNR, 21 versus 11), 30 despite of its thicker membrane and larger nanopore size. We believe the low-noise and high-speed 31 sensing capability of sapphire nanopore chips, together with their scalable fabrication strategy, 32 will find broad use in a number of applications in molecular sensing and beyond. 33

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Single-molecule protein sensing in a nanopore: a tutorial

Cited by 235 publications

References 50 publications

Mapping shifts in nanopore signal to changes in protein and protein-DNA conformation

Mapping shifts in nanopore signal to changes in protein and protein-DNA conformation

A designer FG-Nup that reconstitutes the selective transport barrier of the Nuclear Pore Complex

Sapphire-Supported Nanopores for Low-Noise DNA Sensing

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