Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Quantifying Nanomolar Protein Concentrations Using Designed DNA Carriers and Solid-State Nanopores.

ABSTRACT: Designed "DNA carriers" have been proposed as a new method for nanopore based specific protein detection. In this system, target protein molecules bind to a long DNA strand at a defined position creating a second level transient current drop against the background DNA translocation. Here, we demonstrate the ability of this system to quantify protein concentrations in the nanomolar range. After incubation with target protein at different concentrations, the fraction of DNA translocations showing a secondary current spike allows for the quantification of the corresponding protein concentration. For our proof-of-principle experiments we use two standard binding systems, biotin-streptavidin and digoxigenin-antidigoxigenin, that allow for measurements of the concentration down to the low nanomolar range. The results demonstrate the potential for a novel quantitative and specific protein detection scheme using the DNA carrier method.

SUBMITTER: Kong J

PROVIDER: S-EPMC4901370 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Quantifying Nanomolar Protein Concentrations Using Designed DNA Carriers and Solid-State Nanopores.

Kong Jinglin J Bell Nicholas A W NA Keyser Ulrich F UF

Nano letters 20160503 6

Designed "DNA carriers" have been proposed as a new method for nanopore based specific protein detection. In this system, target protein molecules bind to a long DNA strand at a defined position creating a second level transient current drop against the background DNA translocation. Here, we demonstrate the ability of this system to quantify protein concentrations in the nanomolar range. After incubation with target protein at different concentrations, the fraction of DNA translocations showing ...[more]

PMID: 27121643

Similar Datasets

Quantifying mammalian genomic DNA hydroxymethylcytosine content using solid-state nanopores.

Project description:5-hydroxymethylcytosine (5 hmC), the oxidized form of 5-methylcytosine (5 mC), is a base modification with emerging importance in biology and disease. However, like most epigenetic elements, it is transparent to many conventional genetic techniques and is thus challenging to probe. Here, we report a rapid solid-state nanopore assay that is capable of resolving 5 hmC with high specificity and sensitivity and demonstrate its utility in assessing global modification abundance in genomic DNA.

| S-EPMC4935868 | biostudies-literature

Specific protein detection using designed DNA carriers and nanopores.

Project description:Nanopores are a versatile technique for the detection and characterization of single molecules in solution. An ongoing challenge in the field is to find methods to selectively detect specific biomolecules. In this work we describe a new technique for sensing specific proteins using unmodified solid-state nanopores. We engineered a double strand of DNA by hybridizing nearly two hundred oligonucleotides to a linearized version of the m13mp18 virus genome. This engineered double strand, which we call a DNA carrier, allows positioning of protein binding sites at nanometer accurate intervals along its contour via DNA conjugation chemistry. We measure the ionic current signal of translocating DNA carriers as a function of the number of binding sites and show detection down to the single protein level. Furthermore, we use DNA carriers to develop an assay for identifying a single protein species within a protein mixture.

| S-EPMC4353036 | biostudies-literature

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

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-literature

Genomic Pathogen Typing Using Solid-State Nanopores.

Project description:In clinical settings, rapid and accurate characterization of pathogens is essential for effective treatment of patients; however, subtle genetic changes in pathogens which elude traditional phenotypic typing may confer dangerous pathogenic properties such as toxicity, antibiotic resistance, or virulence. Existing options for molecular typing techniques characterize the critical genomic changes that distinguish harmful and benign strains, yet the well-established approaches, in particular those that rely on electrophoretic separation of nucleic acid fragments on a gel, have room for only incremental future improvements in speed, cost, and complexity. Solid-state nanopores are an emerging class of single-molecule sensors that can electrophoretically characterize charged biopolymers, and which offer significant advantages in terms of sample and reagent requirements, readout speed, parallelization, and automation. We present here the first application of nanopores for single-molecule molecular typing using length based "fingerprints" of critical sites in bacterial genomes. This technique is highly adaptable for detection of different types of genetic variation; as we illustrate using prototypical examples including Mycobacterium tuberculosis and methicillin-resistant Streptococcus aureus, the solid-state nanopore diagnostic platform may be used to detect large insertions or deletions, small insertions or deletions, and even single-nucleotide variations in bacterial DNA. We further show that Bayesian classification of test samples can provide highly confident pathogen typing results based on only a few tens of independent single-molecule events, making this method extremely sensitive and statistically robust.

| S-EPMC4643041 | biostudies-literature

High-bandwidth protein analysis using solid-state nanopores.

Project description:High-bandwidth measurements of the ion current through hafnium oxide and silicon nitride nanopores allow the analysis of sub-30 kD protein molecules with unprecedented time resolution and detection efficiency. Measured capture rates suggest that at moderate transmembrane bias values, a substantial fraction of protein translocation events are detected. Our dwell-time resolution of 2.5 μs enables translocation time distributions to be fit to a first-passage time distribution derived from a 1D diffusion-drift model. The fits yield drift velocities that scale linearly with voltage, consistent with an electrophoretic process. Further, protein diffusion constants (D) are lower than the bulk diffusion constants (D0) by a factor of ~50, and are voltage-independent in the regime tested. We reason that deviations of D from D0 are a result of confinement-driven pore/protein interactions, previously observed in porous systems. A straightforward Kramers model for this inhibited diffusion points to 9- to 12-kJ/mol interactions of the proteins with the nanopore. Reduction of μ and D are found to be material-dependent. Comparison of current-blockage levels of each protein yields volumetric information for the two proteins that is in good agreement with dynamic light scattering measurements. Finally, detection of a protein-protein complex is achieved.

| S-EPMC3944622 | biostudies-literature

Differential Enzyme Flexibility Probed Using Solid-State Nanopores.

Project description:Enzymes and motor proteins are dynamic macromolecules that coexist in a number of conformations of similar energies. Protein function is usually accompanied by a change in structure and flexibility, often induced upon binding to ligands. However, while measuring protein flexibility changes between active and resting states is of therapeutic significance, it remains a challenge. Recently, our group has demonstrated that breadth of signal amplitudes in measured electrical signatures as an ensemble of individual protein molecules is driven through solid-state nanopores and correlates with protein conformational dynamics. Here, we extend our study to resolve subtle flexibility variation in dihydrofolate reductase mutants from unlabeled single molecules in solution. We first demonstrate using a canonical protein system, adenylate kinase, that both size and flexibility changes can be observed upon binding to a substrate that locks the protein in a closed conformation. Next, we investigate the influence of voltage bias and pore geometry on the measured electrical pulse statistics during protein transport. Finally, using the optimal experimental conditions, we systematically study a series of wild-type and mutant dihydrofolate reductase proteins, finding a good correlation between nanopore-measured protein conformational dynamics and equilibrium bulk fluorescence probe measurements. Our results unequivocally demonstrate that nanopore-based measurements reliably probe conformational diversity in native protein ensembles.

| S-EPMC9016714 | 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