The Dynamics of a Molecular Plug Docked onto a Solid-State Nanopore

2018 ◽  
Author(s):  
Xin Shi ◽  
Qiao Li ◽  
Rui Gao ◽  
Wei Si ◽  
Shao-Chuang Liu ◽  
...  
Keyword(s):  
Solid State ◽  
Single Molecule ◽  
Conformational Changes ◽  
Ionic Current ◽  
Observation Time ◽  
Random Coil ◽  
Dna Complexes ◽  
Dna Complex ◽  
Identification And Characterization

<a></a><a>Docking of a protein-DNA complex onto a nanopore can provide ample observation time for a detailed inspection of the complex, enabling collection of biophysical data for detection, identification, and characterization of the biomolecules. While docking of a protein-DNA complex onto a biological nanopore has enabled analytic applications of nanopores including DNA sequencing, the application of the same principle to solid-state nanopores is tempered by poor understanding of the docking process. Here, we elucidate the behaviour of individual protein-DNA complexes docked onto a solid-state nanopore by monitoring the nanopore ionic current. </a><a>Repeat docking of monovalent streptavidin-DNA complexes is found to produce ionic current blockades that fluctuate between discrete levels within the same current blockade. </a>We elucidate the roles of the protein plug and the DNA tether in the docking process, finding the docking configurations to determine the multitude of the current blockade levels whereas the frequency of the current level switching to be determined by the interactions between the molecules and the solid-state membrane. Finally, we prove the feasibility of using the nanopore docking principle for single molecule sensing using solid-state nanopores by detecting conformational changes of a tethered DNA molecule from a random coil to an i-motif states.

The Dynamics of a Molecular Plug Docked onto a Solid-State Nanopore

2018 ◽  
Author(s):  
Xin Shi ◽  
Qiao Li ◽  
Rui Gao ◽  
Wei Si ◽  
Shao-Chuang Liu ◽  
...  
Keyword(s):  
Solid State ◽  
Single Molecule ◽  
Conformational Changes ◽  
Ionic Current ◽  
Observation Time ◽  
Random Coil ◽  
Dna Complexes ◽  
Dna Complex ◽  
Identification And Characterization

<a></a><a>Docking of a protein-DNA complex onto a nanopore can provide ample observation time for a detailed inspection of the complex, enabling collection of biophysical data for detection, identification, and characterization of the biomolecules. While docking of a protein-DNA complex onto a biological nanopore has enabled analytic applications of nanopores including DNA sequencing, the application of the same principle to solid-state nanopores is tempered by poor understanding of the docking process. Here, we elucidate the behaviour of individual protein-DNA complexes docked onto a solid-state nanopore by monitoring the nanopore ionic current. </a><a>Repeat docking of monovalent streptavidin-DNA complexes is found to produce ionic current blockades that fluctuate between discrete levels within the same current blockade. </a>We elucidate the roles of the protein plug and the DNA tether in the docking process, finding the docking configurations to determine the multitude of the current blockade levels whereas the frequency of the current level switching to be determined by the interactions between the molecules and the solid-state membrane. Finally, we prove the feasibility of using the nanopore docking principle for single molecule sensing using solid-state nanopores by detecting conformational changes of a tethered DNA molecule from a random coil to an i-motif states.

scholarly journals Using single-molecule FRET to probe the nucleotide-dependent conformational landscape of polymerase β-DNA complexes

2020 ◽  
Vol 295 (27) ◽  
pp. 9012-9020
Author(s):  
Carel Fijen ◽  
Mariam M. Mahmoud ◽  
Meike Kronenberg ◽  
Rebecca Kaup ◽  
Mattia Fontana ◽  
...  
Keyword(s):  
Dna Repair ◽  
Single Molecule ◽  
Conformational Changes ◽  
Dna Complexes ◽  
Single Molecule Fret ◽  
Nucleotide Incorporation ◽  
Eukaryotic Dna ◽  
Cellular Dna ◽  
Dna Complex ◽  
Gapped Dna

Eukaryotic DNA polymerase β (Pol β) plays an important role in cellular DNA repair, as it fills short gaps in dsDNA that result from removal of damaged bases. Since defects in DNA repair may lead to cancer and genetic instabilities, Pol β has been extensively studied, especially its mechanisms for substrate binding and a fidelity-related conformational change referred to as “fingers closing.” Here, we applied single-molecule FRET to measure distance changes associated with DNA binding and prechemistry fingers movement of human Pol β. First, using a doubly labeled DNA construct, we show that Pol β bends the gapped DNA substrate less than indicated by previously reported crystal structures. Second, using acceptor-labeled Pol β and donor-labeled DNA, we visualized dynamic fingers closing in single Pol β-DNA complexes upon addition of complementary nucleotides and derived rates of conformational changes. We further found that, while incorrect nucleotides are quickly rejected, they nonetheless stabilize the polymerase-DNA complex, suggesting that Pol β, when bound to a lesion, has a strong commitment to nucleotide incorporation and thus repair. In summary, the observation and quantification of fingers movement in human Pol β reported here provide new insights into the delicate mechanisms of prechemistry nucleotide selection.

scholarly journals Comparing current noise in biological and solid-state nanopores

2019 ◽  
Author(s):  
A. Fragasso ◽  
S. Schmid ◽  
C. Dekker
Keyword(s):  
Solid State ◽  
Single Molecule ◽  
Signal To Noise Ratio ◽  
Low Cost ◽  
Ionic Current ◽  
Low Noise ◽  
Signal To Noise ◽  
Experimental Conditions ◽  
High Frequencies ◽  
Noise Ratio

AbstractNanopores bear great potential as single-molecule tools for bioanalytical sensing and sequencing, due to their exceptional sensing capabilities, high-throughput, and low cost. The detection principle relies on detecting small differences in the ionic current as biomolecules traverse the nanopore. A major bottleneck for the further progress of this technology is the noise that is present in the ionic current recordings, because it limits the signal-to-noise ratio and thereby the effective time resolution of the experiment. Here, we review the main types of noise at low and high frequencies and discuss the underlying physics. Moreover, we compare biological and solid-state nanopores in terms of the signal-to-noise ratio (SNR), the important figure of merit, by measuring free translocations of a short ssDNA through a selected set of nanopores under typical experimental conditions. We find that SiNx solid-state nanopores provide the highest SNR, due to the large currents at which they can be operated and the relatively low noise at high frequencies. However, the real game-changer for many applications is a controlled slowdown of the translocation speed, which for MspA was shown to increase the SNR >160-fold. Finally, we discuss practical approaches for lowering the noise for optimal experimental performance and further development of the nanopore technology.

scholarly journals A Solid-State Nanopore-Based Single-Molecule Approach for Label-free Characterization of Plant Polysaccharides

2020 ◽  
pp. 100106
Author(s):  
Yao Cai ◽  
Baocai Zhang ◽  
Liyuan Liang ◽  
Sen Wang ◽  
Lanjun Zhang ◽  
...  
Keyword(s):  
Solid State ◽  
Single Molecule ◽  
Label Free ◽  
Plant Polysaccharides

Identification and Characterization of Solid-State Nature of 2-Chloromandelic Acid

2009 ◽  
Vol 98 (5) ◽  
pp. 1835-1844 ◽  
Author(s):  
Quan He ◽  
Jesse Zhu ◽  
Hassan Gomaa ◽  
Michael Jennings ◽  
Sohrab Rohani
Keyword(s):  
Solid State ◽  
Identification And Characterization ◽  
State Nature

scholarly journals Single-molecule FRET dynamics of molecular motors in an ABEL Trap

2020 ◽  
Author(s):  
Maria Dienerowitz ◽  
Jamieson A. L. Howard ◽  
Steven D. Quinn ◽  
Frank Dienerowitz ◽  
Mark C. Leake
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Conformational Changes ◽  
Molecular Motors ◽  
Rational Design ◽  
Molecular Motor ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Observation Time

AbstractSingle-molecule Förster resonance energy transfer (smFRET) of molecular motors provides transformative insights into their dynamics and conformational changes both at high temporal and spatial resolution simultaneously. However, a key challenge of such FRET investigations is to observe a molecule in action for long enough without restricting its natural function. The Anti-Brownian ELectrokinetic Trap (ABEL trap) sets out to combine smFRET with molecular confinement to enable observation times of up to several seconds while removing any requirement of tethered surface attachment of the molecule in question. In addition, the ABEL trap’s inherent ability to selectively capture FRET active molecules accelerates the data acquisition process. Here we exemplify the capabilities of the ABEL trap in performing extended timescale smFRET measurements on the molecular motor Rep, which is crucial for removing protein blocks ahead of the advancing DNA replication machinery and for restarting stalled DNA replication. We are able to monitor single Rep molecules up to 6 s with 1 ms time resolution capturing multiple conformational switching events during the observation time. Here we provide a step-by-step guide for the rational design, construction and implementation of the ABEL trap for smFRET detection of Rep in vitro. We include details of how to model the electric potential at the trap site and use Hidden Markov analysis of the smFRET trajectories.

DNA polymerase activity at the single-molecule level

2011 ◽  
Vol 39 (2) ◽  
pp. 595-599 ◽  
Author(s):  
Joshua P. Gill ◽  
Jun Wang ◽  
David P. Millar
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Protein Complexes ◽  
Dna Polymerases ◽  
Polymerase Activity ◽  
Single Molecule Level ◽  
Nucleotide Incorporation ◽  
Single Molecule Fluorescence Spectroscopy ◽  
Conformational Rearrangements ◽  
Dna Complex

DNA polymerases are essential enzymes responsible for replication and repair of DNA in all organisms. To replicate DNA with high fidelity, DNA polymerases must select the correct incoming nucleotide substrate during each cycle of nucleotide incorporation, in accordance with the templating base. When an incorrect nucleotide is sometimes inserted, the polymerase uses a separate 3′→5′ exonuclease to remove the misincorporated base (proofreading). Large conformational rearrangements of the polymerase–DNA complex occur during both the nucleotide incorporation and proofreading steps. Single-molecule fluorescence spectroscopy provides a unique tool for observation of these dynamic conformational changes in real-time, without the need to synchronize a population of DNA–protein complexes.

scholarly journals Field effect control of translocation dynamics in surround-gate nanopores

2021 ◽  
Vol 2 (1) ◽  
Author(s):  
Makusu Tsutsui ◽  
Sou Ryuzaki ◽  
Kazumichi Yokota ◽  
Yuhui He ◽  
Takashi Washio ◽  
...  
Keyword(s):  
Solid State ◽  
Water Flow ◽  
Single Molecule ◽  
Ionic Current ◽  
Critical Issue ◽  
Hydrodynamic Drag ◽  
Single Particles ◽  
Drag Forces ◽  
Fine Control ◽  
Controllable Motion

AbstractControlling the fast electrophoresis of nano-objects in solid-state nanopores is a critical issue for achieving electrical analysis of single-particles by ionic current. In particular, it is crucial to slow-down the translocation dynamics of nanoparticles. We herein report that a focused electric field and associated water flow in a surround-gate nanopore can be used to trap and manipulate a nanoscale object. We fine-control the electroosmosis-induced water flow by modulating the wall surface potential via gate voltage. We find that a nanoparticle can be captured in the vicinity of the conduit by balancing the counteracting electrophoretic and hydrodynamic drag forces. By creating a subtle force imbalance, in addition, we also demonstrate a gate-controllable motion of single-particles moving at an extremely slow speed of several tens of nanometers per second. The present method may be useful in single-molecule detection by solid-state nanopores and nanochannels.

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