Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Detection of CRISPR-dCas9 on DNA with Solid-State Nanopores.

ABSTRACT: Solid-state nanopores have emerged as promising platforms for biosensing including diagnostics for disease detection. Here we show nanopore experiments that detect CRISPR-dCas9, a sequence-specific RNA-guided protein system that specifically binds to a target DNA sequence. While CRISPR-Cas9 is acclaimed for its gene editing potential, the CRISPR-dCas9 variant employed here does not cut DNA but instead remains tightly bound at a user-defined binding site, thus providing an excellent target for biosensing. In our nanopore experiments, we observe the CRISPR-dCas9 proteins as local spikes that appear on top of the ionic current blockade signal of DNA molecules that translocate through the nanopore. The proteins exhibit a pronounced blockade signal that allows for facile identification of the targeted sequence. Even at the high salt conditions (1 M LiCl) required for nanopore experiments, dCas9 proteins are found to remain stably bound. The binding position of the target sequence can be read from the spike position along the DNA signal. We anticipate applications of this nanopore-based CRISPR-dCas9 biosensing approach in DNA-typing based diagnostics such as quick disease-strain identification, antibiotic-resistance detection, and genome typing.

SUBMITTER: Yang W

PROVIDER: S-EPMC6187524 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Detection of DNA hybridizations using solid-state nanopores.

Project description:We report an experimental study of using DNA translocation through solid-state nanopores to detect the sequential arrangement of two double-stranded 12-mer hybridization segments on a single-stranded DNA molecule. The sample DNA is a trimer molecule formed by hybridizing three single-stranded oligonucleotides. A polystyrene bead is attached to the end of the trimer DNA, providing a mechanism in slowing down the translocation and suppressing the thermal diffusion, thereby allowing the detection of short features of DNA by standard patch-clamp electronics. The electrical signature of the translocation of a trimer molecule through a nanopore has been identified successfully in the temporal traces of ionic current. The results reported here represent the first successful attempt in using a solid-state nanopore as an ionic scanning device in resolving individual hybridization segments (or 'probes') on a DNA molecule.

| S-EPMC4811674 | biostudies-literature

Detection of SARS-CoV-2 with Solid-State CRISPR-Cas12a-Assisted Nanopores.

Project description:The outbreak of the SARS-CoV-2 caused the disease COVID-19 to spread globally. Specific and sensitive detection of SARS-CoV-2 facilitates early intervention and prevents the disease from spreading. Here, we present a solid-state CRISPR-Cas12a-assisted nanopore (SCAN) sensing strategy for the specific detection of SARS-CoV-2. We introduced a nanopore-sized counting method to measure the cleavage ratio of reporters, which is used as a criterion for positive/negative classification. A kinetic cleavage model was developed and validated to predict the reporter size distributions. The model revealed the trade-offs between sensitivity, turnaround time, and false-positive rate of the SARS-CoV-2 SCAN. With preamplification and a 30 min CRISPR Cas12a assay, we achieved excellent specificity against other common human coronaviruses and a limit of detection of 13.5 copies/μL (22.5 aM) of viral RNA at a confidence level of 95%. These results suggested that the SCAN could provide a rapid, sensitive, and specific analysis of SARS-CoV-2.

| S-EPMC8491552 | biostudies-literature

DNA profiling using solid-state nanopores: detection of DNA-binding molecules.

Project description:We present a novel single-molecule method for rapidly evaluating small-molecule binding to individual DNA molecules using nanopores fabricated in ultrathin silicon membranes. A measurable shift in the residual ion current through a approximately 3.5 nm pore results from threading of a dye-intercalated DNA molecule, as compared to the typical residual current of native DNA. The average level of the residual current can be used to directly quantify the fraction of bound molecules to DNA, providing a new way to determine binding isotherms. Spatial sensitivity is also demonstrated by designing a two-segment DNA molecule that contains small-molecule binding sites in one of its two segments. Translocations of such molecules exhibit two current levels upon incubation with a DNA-binding dye, caused by selectively bound dye in one of the DNA segments. Our results, as shown here with four different dyes, coincide well with bulk fluorescence measurements performed under identical conditions. The nanopore approach for "reading-out" molecular binding along a DNA molecule, combined with the miniscule amounts of DNA required and the potential for scalability using nanopore arrays, provide a novel platform for future applications in analytical drug screening.

| S-EPMC2871189 | biostudies-literature

Detection and quantification of methylation in DNA using solid-state nanopores.

Project description:Epigenetic modifications in eukaryotic genomes occur primarily in the form of 5-methylcytosine (5 mC). These modifications are heavily involved in transcriptional repression, gene regulation, development and the progression of diseases including cancer. We report a new single-molecule assay for the detection of DNA methylation using solid-state nanopores. Methylation is detected by selectively labeling methylation sites with MBD1 (MBD-1x) proteins, the complex inducing a 3 fold increase in ionic blockage current relative to unmethylated DNA. Furthermore, the discrimination of methylated and unmethylated DNA is demonstrated in the presence of only a single bound protein, thereby giving a resolution of a single methylated CpG dinucleotide. The extent of methylation of a target molecule could also be coarsely quantified using this novel approach. This nanopore-based methylation sensitive assay circumvents the need for bisulfite conversion, fluorescent labeling, and PCR and could therefore prove very useful in studying the role of epigenetics in human disease.

| S-EPMC3593219 | biostudies-literature

Detecting DNA depurination with solid-state nanopores.

Project description:Among the different types of DNA damage that occur endogenously in the cell, depurination is especially prevalent. These lesions can initiate mutagenesis and have been implicated in a variety of diseases, including cancer. Here, we demonstrate a new approach for the detection of depurination at the single-molecule scale using solid-state nanopores. We induce depurination in short duplex DNA using acidic conditions and observe that the presence of apurinic sites results in significantly slower dynamics during electrokinetic translocation. This procedure may be valuable as a diagnostic for in situ quantification of DNA depurination.

| S-EPMC4079296 | biostudies-literature

DNA translocations through solid-state plasmonic nanopores.

Project description:Nanopores enable label-free detection and analysis of single biomolecules. Here, we investigate DNA translocations through a novel type of plasmonic nanopore based on a gold bowtie nanoantenna with a solid-state nanopore at the plasmonic hot spot. Plasmonic excitation of the nanopore is found to influence both the sensor signal (nanopore ionic conductance blockade during DNA translocation) and the process that captures DNA into the nanopore, without affecting the duration time of the translocations. Most striking is a strong plasmon-induced enhancement of the rate of DNA translocation events in lithium chloride (LiCl, already 10-fold enhancement at a few mW of laser power). This provides a means to utilize the excellent spatiotemporal resolution of DNA interrogations with nanopores in LiCl buffers, which is known to suffer from low event rates. We propose a mechanism based on plasmon-induced local heating and thermophoresis as explanation of our observations.

| S-EPMC4264857 | biostudies-other

Toward detection of DNA-bound proteins using solid-state nanopores: insights from computer simulations.

Project description:Through all-atom molecular dynamics simulations, we explore the use of nanopores in thin synthetic membranes for detection and identification of DNA binding proteins. Reproducing the setup of a typical experiment, we simulate electric field driven transport of DNA-bound proteins through nanopores smaller in diameter than the proteins. As model systems, we use restriction enzymes EcoRI and BamHI specifically and nonspecifically bound to a fragment of dsDNA, and streptavidin and NeutrAvidin proteins bound to dsDNA and ssDNA via a biotin linker. Our simulations elucidate the molecular mechanics of nanopore-induced rupture of a protein-DNA complex, the effective force applied to the DNA-protein bond by the electrophoretic force in a nanopore, and the role of DNA-surface interactions in the rupture process. We evaluate the ability of the nanopore ionic current and the local electrostatic potential measured by an embedded electrode to report capture of DNA, capture of a DNA-bound protein, and rupture of the DNA-protein bond. We find that changes in the strain on dsDNA can reveal the rupture of a protein-DNA complex by altering both the nanopore ionic current and the potential of the embedded electrode. Based on the results of our simulations, we suggest a new method for detection of DNA binding proteins that utilizes peeling of a nicked double strand under the electrophoretic force in a nanopore.

| S-EPMC3789251 | biostudies-literature

Temperature dependence of DNA translocations through solid-state nanopores.

Project description:In order to gain a better physical understanding of DNA translocations through solid-state nanopores, we study the temperature dependence of ?-DNA translocations through 10 nm diameter silicon nitride nanopores, both experimentally and theoretically. The measured ionic conductance G, the DNA-induced ionic-conductance blockades [Formula: see text] and the event frequency ? all increase with increasing temperature while the DNA translocation time ? decreases. G and [Formula: see text] are accurately described when bulk and surface conductances of the nanopore are considered and access resistance is incorporated appropriately. Viscous drag on the untranslocated part of the DNA coil is found to dominate the temperature dependence of the translocation times and the event rate is well described by a balance between diffusion and electrophoretic motion. The good fit between modeled and measured properties of DNA translocations through solid-state nanopores in this first comprehensive temperature study, suggest that our model captures the relevant physics of the process.

| S-EPMC4503867 | biostudies-literature

DNA translocation governed by interactions with solid-state nanopores.

Project description:We investigate the voltage-driven translocation dynamics of individual DNA molecules through solid-state nanopores in the diameter range 2.7-5 nm. Our studies reveal an order of magnitude increase in the translocation times when the pore diameter is decreased from 5 to 2.7 nm, and steep temperature dependence, nearly threefold larger than would be expected if the dynamics were governed by viscous drag. As previously predicted for an interaction-dominated translocation process, we observe exponential voltage dependence on translocation times. Mean translocation times scale with DNA length by two power laws: for short DNA molecules, in the range 150-3500 bp, we find an exponent of 1.40, whereas for longer molecules, an exponent of 2.28 dominates. Surprisingly, we find a transition in the fraction of ion current blocked by DNA, from a length-independent regime for short DNA molecules to a regime where the longer the DNA, the more current is blocked. Temperature dependence studies reveal that for increasing DNA lengths, additional interactions are responsible for the slower DNA dynamics. Our results can be rationalized by considering DNA/pore interactions as the predominant factor determining DNA translocation dynamics in small pores. These interactions markedly slow down the translocation rate, enabling higher temporal resolution than observed with larger pores. These findings shed light on the transport properties of DNA in small pores, relevant for future nanopore applications, such as DNA sequencing and genotyping.

| S-EPMC2576395 | biostudies-literature

Noise in solid-state nanopores.

Project description:We study ionic current fluctuations in solid-state nanopores over a wide frequency range and present a complete description of the noise characteristics. At low frequencies (f approximately < 100 Hz) we observe 1/f-type of noise. We analyze this low-frequency noise at different salt concentrations and find that the noise power remarkably scales linearly with the inverse number of charge carriers, in agreement with Hooge's relation. We find a Hooge parameter alpha = (1.1 +/- 0.1) x 10(-4). In the high-frequency regime (f approximately > 1 kHz), we can model the increase in current power spectral density with frequency through a calculation of the Johnson noise. Finally, we use these results to compute the signal-to-noise ratio for DNA translocation for different salt concentrations and nanopore diameters, yielding the parameters for optimal detection efficiency.

| S-EPMC2206550 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data