Translocation of RecA-Coated Double-Stranded DNA through Solid-State Nanopores

Smeets, Ralph M. M.; Kowalczyk, Stefan W.; Hall, Adam R.; Dekker, Nynke H.; Dekker, Cees

doi:10.1021/nl803189k

Cited by 123 publications

(136 citation statements)

References 34 publications

Supporting

Mentioning

129

Contrasting

Order By: Relevance

“…The possibility of applying nanopores to the analysis of nucleic acids, in particular DNA sequencing, has generated interest 3 , and motivated fundamental studies of the physics of nanopore translocations [4][5][6][7][8][9][10][11][12][13][14][15][16][17] . The uses of solid-state nanopores have recently expanded to include detecting single proteins 18 , mapping structural features along RecA-bound DNA-protein complexes 19 and detecting spherical and icosahedral virus strains [20][21][22] . Such advances underline the importance of expanding our understanding of nanopore translocations beyond the case of DNA.…”

mentioning

confidence: 99%

Stiff filamentous virus translocations through solid-state nanopores

et al. 2014

View full text Add to dashboard Cite

The ionic conductance through a nanometer-sized pore in a membrane changes when a biopolymer slides through it, making nanopores sensitive to single molecules in solution. Their possible use for sequencing has motivated numerous studies on how DNA, a semiflexible polymer, translocates nanopores. Here we study voltage-driven dynamics of the stiff filamentous virus fd with experiments and simulations to investigate the basic physics of polymer translocations. We find that the electric field distribution aligns an approaching fd with the nanopore, promoting its capture, but it also pulls fd sideways against the membrane after failed translocation attempts until thermal fluctuations reorient the virus for translocation. fd is too stiff to translocate in folded configurations. It therefore translocates linearly, exhibiting a voltage-independent mobility and obeying first-passage-time statistics. Surprisingly, lengthwise Brownian motion only partially accounts for the translocation velocity fluctuations. We also observe a voltage-dependent contribution whose origin is only partially determined.

show abstract

mentioning

confidence: 99%

Stiff filamentous virus translocations through solid-state nanopores

et al. 2014

View full text Add to dashboard Cite

show abstract

“…Studies by Kowalczyk on RecA bound DNA through large ~30nm nanopores confirmed the detection of two distinct blockade levels corresponding to regions of RecA coated DNA and uncoated DNA. [220,221] RecA has a molecular weight (~38 kDa) and cross sectional diameter (~7 nm) that is similar to MBD2. Thus, distinct current signatures from MBD2 bound DNA are expected relative to native DNA in ~15 nm Al 2 O 3 nanopores.…”

Section: Analysis Of Mbd2 Bound Methylated Dna Using Electrical Currementioning

confidence: 99%

Solid-State Nanopore Sensors for Nucleic Acid Analysis

Venkatesan

Bashir

2011

Nanopores

View full text Add to dashboard Cite

Nanopore DNA analysis is an emerging technique that involves electrophoretically driving DNA molecules through a nano-scale pore in solution and monitoring the corresponding change in ionic pore current. This versatile approach permits the label-free, amplification-free analysis of charged polymers (single stranded DNA, double stranded DNA and RNA) ranging in length from single nucleotides to kilobase long genomic DNA fragments with subnanometer resolution.Recent advances in nanopores suggest that this low-cost, highly scalable technology could lend itself to the development of third generation DNA sequencing technologies, promising rapid and reliable sequencing of the human diploid genome for under $1000.Here, we report the development of versatile, nano-manufactured Al 2 O 3 solid-state nanopores and nanopore arrays for rapid, label-free, single-molecule detection and analysis of DNA and protein. This nano-scale technology has proven to be reliable, affordable, and mass producible, and allows for integration with VLSI processes. A detailed characterization of nanopore performance in terms of electrical noise, mechanical robustness and materials analysis is provided, and the functionality of this technology in experimental DNA biophysics is explored.A framework for the application of this technology to medical diagnostics and sequencing is also presented. Specifically, studies involved the detection of DNA-protein complexes, a viable strategy in screening methylation patterns in panels of genes for early cancer detection, and the creation of lipid bilayer coated nanopore sensors, useful in creating hybrid biological/solid-state nanopores for DNA sequencing applications.The concept of a gated nanopore is also presented with preliminary results. The fabrication of this novel system has been enabled by the recent discovery of graphene, a highly versatile material with remarkable electrical and mechanical properties. Direct modulation of the nanopore conductance was observed through the application of potentials to the graphene gate.These exciting results suggest this technology could potentially be useful in slowing down or trapping a DNA molecule in the pore, thereby enabling solid-state nanopore sequencing.iii

show abstract

“…54 Due to positive cooperativity RecA forms long filaments on DNA extended by 50% comparing to the DNA molecule. 16 The linear charge density of such filaments is approximately twice higher than those for the bare DNA. 15 In accordance with theory, the electrophoretic force on RecAcoated DNA inside nanopores and measured with optical tweezers was 2−4 times higher compared to the DNA.…”

mentioning

confidence: 95%

Relevance of the Drag Force during Controlled Translocation of a DNA–Protein Complex through a Glass Nanocapillary

2015

View full text Add to dashboard Cite

Combination of glass nanocapillaries with optical tweezers allowed us to detect DNA−protein complexes in physiological conditions. In this system, a protein bound to DNA is characterized by a simultaneous change of the force and ionic current signals from the level observed for the bare DNA. Controlled displacement of the protein away from the nanocapillary opening revealed decay in the values of the force and ionic current. Negatively charged proteins EcoRI, RecA, and RNA polymerase formed complexes with DNA that experienced electrophoretic force lower than the bare DNA inside nanocapillaries. Force profiles obtained for DNA-RecA in our system were different than those in the system with nanopores in membranes and optical tweezers. We suggest that such behavior is due to the dominant impact of the drag force comparing to the electrostatic force acting on a DNA−protein complex inside nanocapillaries. We explained our results using a stochastic model taking into account the conical shape of glass nanocapillaries.

show abstract

Translocation of RecA-Coated Double-Stranded DNA through Solid-State Nanopores

Cited by 123 publications

References 34 publications

Stiff filamentous virus translocations through solid-state nanopores

Stiff filamentous virus translocations through solid-state nanopores

Solid-State Nanopore Sensors for Nucleic Acid Analysis

Relevance of the Drag Force during Controlled Translocation of a DNA–Protein Complex through a Glass Nanocapillary

Contact Info

Product

Resources

About