Browse
Submit Data
Databases
API
Help

Dataset Information

19 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Direct Detection of Unnatural DNA Nucleotides dNaM and d5SICS using the MspA Nanopore.

ABSTRACT: Malyshev et al. showed that the four-letter genetic code within a living organism could be expanded to include the unnatural DNA bases dNaM and d5SICS. However, verification and detection of these unnatural bases in DNA requires new sequencing techniques. Here we provide proof of concept detection of dNaM and d5SICS in DNA oligomers via nanopore sequencing using the nanopore MspA. We find that both phi29 DNA polymerase and Hel308 helicase are capable of controlling the motion of DNA containing dNaM and d5SICS through the pore and that single reads are sufficient to detect the presence and location of dNaM and d5SICS within single molecules.

SUBMITTER: Craig JM

PROVIDER: S-EPMC4654578 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Direct Detection of Unnatural DNA Nucleotides dNaM and d5SICS using the MspA Nanopore.

Craig Jonathan M JM Laszlo Andrew H AH Derrington Ian M IM Ross Brian C BC Brinkerhoff Henry H Nova Ian C IC Doering Kenji K Tickman Benjamin I BI Svet Mark T MT Gundlach Jens H JH

PloS one 20151120 11

Malyshev et al. showed that the four-letter genetic code within a living organism could be expanded to include the unnatural DNA bases dNaM and d5SICS. However, verification and detection of these unnatural bases in DNA requires new sequencing techniques. Here we provide proof of concept detection of dNaM and d5SICS in DNA oligomers via nanopore sequencing using the nanopore MspA. We find that both phi29 DNA polymerase and Hel308 helicase are capable of controlling the motion of DNA containing d ...[more]

PMID: 26588074

Similar Datasets

Single-molecule DNA detection with an engineered MspA protein nanopore.

Project description:Nanopores hold great promise as single-molecule analytical devices and biophysical model systems because the ionic current blockades they produce contain information about the identity, concentration, structure, and dynamics of target molecules. The porin MspA of Mycobacterium smegmatis has remarkable stability against environmental stresses and can be rationally modified based on its crystal structure. Further, MspA has a short and narrow channel constriction that is promising for DNA sequencing because it may enable improved characterization of short segments of a ssDNA molecule that is threaded through the pore. By eliminating the negative charge in the channel constriction, we designed and constructed an MspA mutant capable of electronically detecting and characterizing single molecules of ssDNA as they are electrophoretically driven through the pore. A second mutant with additional exchanges of negatively-charged residues for positively-charged residues in the vestibule region exhibited a factor of approximately 20 higher interaction rates, required only half as much voltage to observe interaction, and allowed ssDNA to reside in the vestibule approximately 100 times longer than the first mutant. Our results introduce MspA as a nanopore for nucleic acid analysis and highlight its potential as an engineerable platform for single-molecule detection and characterization applications.

| S-EPMC2634888 | biostudies-literature

Nanopore DNA sequencing with MspA.

Project description:Nanopore sequencing has the potential to become a direct, fast, and inexpensive DNA sequencing technology. The simplest form of nanopore DNA sequencing utilizes the hypothesis that individual nucleotides of single-stranded DNA passing through a nanopore will uniquely modulate an ionic current flowing through the pore, allowing the record of the current to yield the DNA sequence. We demonstrate that the ionic current through the engineered Mycobacterium smegmatis porin A, MspA, has the ability to distinguish all four DNA nucleotides and resolve single-nucleotides in single-stranded DNA when double-stranded DNA temporarily holds the nucleotides in the pore constriction. Passing DNA with a series of double-stranded sections through MspA provides proof of principle of a simple DNA sequencing method using a nanopore. These findings highlight the importance of MspA in the future of nanopore sequencing.

| S-EPMC2941267 | biostudies-literature

Synthesized DNA with unnatural nucleotides

Project description:Synthesized DNA with unnatural nucleotides Synthetic Genomics

| PRJNA89669 | ENA

Nucleotide discrimination with DNA immobilized in the MspA nanopore.

Project description:Nanopore sequencing has the potential to become a fast and low-cost DNA sequencing platform. An ionic current passing through a small pore would directly map the sequence of single stranded DNA (ssDNA) driven through the constriction. The pore protein, MspA, derived from Mycobacterium smegmatis, has a short and narrow channel constriction ideally suited for nanopore sequencing. To study MspA's ability to resolve nucleotides, we held ssDNA within the pore using a biotin-NeutrAvidin complex. We show that homopolymers of adenine, cytosine, thymine, and guanine in MspA exhibit much larger current differences than in α-hemolysin. Additionally, methylated cytosine is distinguishable from unmethylated cytosine. We establish that single nucleotide substitutions within homopolymer ssDNA can be detected when held in MspA's constriction. Using genomic single nucleotide polymorphisms, we demonstrate that single nucleotides within random DNA can be identified. Our results indicate that MspA has high signal-to-noise ratio and the single nucleotide sensitivity desired for nanopore sequencing devices.

| S-EPMC3186796 | biostudies-literature

Therminator DNA Polymerase: Modified Nucleotides and Unnatural Substrates.

Project description:A variant of 9°N DNA polymerase [Genbank ID (AAA88769.1)] with three mutations (D141A, E143A, A485L) and commercialized under the name "Therminator DNA polymerase" has the ability to incorporate a variety of modified nucleotide classes. This Review focuses on how Therminator DNA Polymerase has enabled new technologies in synthetic biology and DNA sequencing. In addition, we discuss mechanisms for increased modified nucleotide incorporation.

| S-EPMC6491775 | biostudies-literature

PEG-labeled nucleotides and nanopore detection for single molecule DNA sequencing by synthesis.

Project description:We describe a novel single molecule nanopore-based sequencing by synthesis (Nano-SBS) strategy that can accurately distinguish four bases by detecting 4 different sized tags released from 5'-phosphate-modified nucleotides. The basic principle is as follows. As each nucleotide is incorporated into the growing DNA strand during the polymerase reaction, its tag is released and enters a nanopore in release order. This produces a unique ionic current blockade signature due to the tag's distinct chemical structure, thereby determining DNA sequence electronically at single molecule level with single base resolution. As proof of principle, we attached four different length PEG-coumarin tags to the terminal phosphate of 2'-deoxyguanosine-5'-tetraphosphate. We demonstrate efficient, accurate incorporation of the nucleotide analogs during the polymerase reaction, and excellent discrimination among the four tags based on nanopore ionic currents. This approach coupled with polymerase attached to the nanopores in an array format should yield a single-molecule electronic Nano-SBS platform.

| S-EPMC3448304 | biostudies-literature

Optical recognition of converted DNA nucleotides for single-molecule DNA sequencing using nanopore arrays.

Project description:We demonstrate the feasibility of a nanopore based single-molecule DNA sequencing method, which employs multicolor readout. Target DNA is converted according to a binary code, which is recognized by molecular beacons with two types of fluorophores. Solid-state nanopores are then used to sequentially strip off the beacons, leading to a series of detectable photon bursts, at high speed. We show that signals from multiple nanopores can be detected simultaneously, allowing straightforward parallelization to large nanopore arrays.

| S-EPMC2883017 | biostudies-literature

Detection and mapping of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore MspA.

Project description:Precise and efficient mapping of epigenetic markers on DNA may become an important clinical tool for prediction and identification of ailments. Methylated CpG sites are involved in gene expression and are biomarkers for diseases such as cancer. Here, we use the engineered biological protein pore Mycobacterium smegmatis porin A (MspA) to detect and map 5-methylcytosine and 5-hydroxymethylcytosine within single strands of DNA. In this unique single-molecule tool, a phi29 DNA polymerase draws ssDNA through the pore in single-nucleotide steps, and the ion current through the pore is recorded. Comparing current levels generated with DNA containing methylated CpG sites to current levels obtained with unmethylated copies of the DNA reveals the precise location of methylated CpG sites. Hydroxymethylation is distinct from methylation and can also be mapped. With a single read, the detection efficiency in a quasirandom DNA strand is 97.5 ± 0.7% for methylation and 97 ± 0.9% for hydroxymethylation.

| S-EPMC3839702 | biostudies-literature

Nanopore-based identification of individual nucleotides for direct RNA sequencing.

Project description:We describe a label-free ribobase identification method, which uses ionic current measurement to resolve ribonucleoside monophosphates or diphosphates in ?-hemolysin protein nanopores containing amino-cyclodextrin adapters. The accuracy of base identification is further investigated through the use of a guanidino-modified adapter. On the basis of these findings, an exosequencing approach is envisioned in which a processive exoribonuclease (polynucleotide phosphorylase) presents sequentially cleaved ribonucleoside diphosphates to a nanopore.

| S-EPMC3899427 | biostudies-literature

Investigating asymmetric salt profiles for nanopore DNA sequencing with biological porin MspA.

Project description:Nanopore DNA sequencing is a promising single-molecule analysis technology. This technique relies on a DNA motor enzyme to control movement of DNA precisely through a nanopore. Specific experimental buffer conditions are required based on the preferred operating conditions of the DNA motor enzyme. While many DNA motor enzymes typically operate in salt concentrations under 100 mM, salt concentration simultaneously affects signal and noise magnitude as well as DNA capture rate in nanopore sequencing, limiting standard experimental conditions to salt concentrations greater than ~100 mM in order to maintain adequate resolution and experimental throughput. We evaluated the signal contribution from ions on both sides of the membrane (cis and trans) by varying cis and trans [KCl] independently during phi29 DNA Polymerase-controlled translocation of DNA through the biological porin MspA. Our studies reveal that during DNA translocation, the negatively charged DNA increases cation selectivity through MspA with the majority of current produced by the flow of K+ ions from trans to cis. Varying trans [K+] has dramatic effects on the signal magnitude, whereas changing cis [Cl-] produces only small effects. Good signal-to-noise can be maintained with cis [Cl-] as small as 20 mM, if the concentration of KCl on the trans side is kept high. These results demonstrate the potential of using salt-sensitive motor enzymes (helicases, polymerases, recombinases) in nanopore systems and offer a guide for selecting buffer conditions in future experiments to simultaneously optimize signal, throughput, and enzyme activity.

| S-EPMC5531483 | 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