Nanopore DNA Research Articles

Nanopore-Based DNA Hard Drives for Rewritable and Secure Data Storage.

Nanopores are powerful single-molecule tools for label-free sensing of nanoscale molecules including DNA that can be used for building designed nanostructures and performing computations. Here, DNA hard drives (DNA-HDs) are introduced based on DNA nanotechnology and nanopore sensing as a rewritable molecular memory system, allowing for storing, operating, and reading data in the changeable three-dimensional structure of DNA. Writing and erasing data are significantly improved compared to previous molecular storage systems by employing controllable attachment and removal of molecules on a long double-stranded DNA. Data reading is achieved by detecting the single molecules at the millisecond time scale using nanopores. The DNA-HD also ensures secure data storage where the data can only be read after providing the correct physical molecular keys. Our approach allows for easy-writing and easy-reading, rewritable, and secure data storage toward a promising miniature scale integration for molecular data storage and computation.

Crowding-Induced DNA Translocation through a Protein Nanopore.

A crowded cellular environment is highly associated with many significant biological processes. However, the effect of molecular crowding on the translocation behavior of DNA through a pore has not been explored. Here, we use nanopore single-molecule analytical technique to quantify the thermodynamics and kinetics of DNA transport under heterogeneous cosolute PEGs. The results demonstrate that the frequency of the translocation event exhibits a nonmonotonic dependence on the crowding agent size, while both the event frequency and translocation time increase monotonically with increasing crowder concentration. In the presence of PEGs, the rate of DNA capture into the nanopore elevates 118.27-fold, and at the same time the translocation velocity decreases from 20 to 120 μs/base. Interestingly, the impact of PEG 4k on the DNA-nanopore interaction is the most notable, with up to ΔΔG = 16.27 kJ mol-1 change in free energy and 764.50-fold increase in the binding constant at concentration of 40% (w/v). The molecular crowding effect will has broad applications in nanopore biosensing and nanopore DNA sequencing in which the strategy to capture analyte and to control the transport is urgently required.

Evaluating the genome and resistome of extensively drug-resistant Klebsiella pneumoniae using native DNA and RNA Nanopore sequencing.

Background Klebsiella pneumoniae frequently harbours multidrug resistance, and current diagnostics struggle to rapidly identify appropriate antibiotics to treat these bacterial infections. The MinION device can sequence native DNA and RNA in real time, providing an opportunity to compare the utility of DNA and RNA for prediction of antibiotic susceptibility. However, the effectiveness of bacterial direct RNA sequencing and base-calling has not previously been investigated. This study interrogated the genome and transcriptome of 4 extensively drug-resistant (XDR) K. pneumoniae clinical isolates; however, further antimicrobial susceptibility testing identified 3 isolates as pandrug-resistant (PDR).ResultsThe majority of acquired resistance (≥75%) resided on plasmids including several megaplasmids (≥100 kb). DNA sequencing detected most resistance genes (≥70%) within 2 hours of sequencing. Neural network–based base-calling of direct RNA achieved up to 86% identity rate, although ≤23% of reads could be aligned. Direct RNA sequencing (with ∼6 times slower pore translocation) was able to identify (within 10 hours) ≥35% of resistance genes, including those associated with resistance to aminoglycosides, β-lactams, trimethoprim, and sulphonamide and also quinolones, rifampicin, fosfomycin, and phenicol in some isolates. Direct RNA sequencing also identified the presence of operons containing up to 3 resistance genes. Polymyxin-resistant isolates showed a heightened transcription of phoPQ (≥2-fold) and the pmrHFIJKLM operon (≥8-fold). Expression levels estimated from direct RNA sequencing displayed strong correlation (Pearson: 0.86) compared to quantitative real-time PCR across 11 resistance genes.ConclusionOverall, MinION sequencing rapidly detected the XDR/PDR K. pneumoniae resistome, and direct RNA sequencing provided accurate estimation of expression levels of these genes.

Recent Progress in Solid-State Nanopore DNA Sequencing

Solid-state nanopores offer a novel platform for single molecule analysis and characterization. In particular, they have attracted significant attention as tools for high-throughput, robust, and low-error DNA sequencing. The successful implementation of solid-state nanopores in emerging third-generation DNA sequencing applications is contingent upon developing methods for scalable fabrication, high-accuracy output, and integration with low-noise electronic architectures. In this presentation, I will cover recent progress our group has made in the production, characterization, and integration of these devices. New high-resolution methods for fabricating nanopores down to the single-atom level (0.5 nm) - ion beam irradiation, photo-oxidation, and controlled chemical etching - will first be discussed. I will then describe recent successes in incorporating solid-state nanopores with high-frequency (10 MHz), low-noise amplifiers and subsequent advances in signal processing. Lastly, a brief perspective on the outlook of solid-state nanopores as well as ongoing work with ion-channel-like pores in two-dimensional (2D) materials will be provided.

Molecular Dynamics Simulation Study of Transverse and Longitudinal Ionic Currents in Solid-State Nanopore DNA Sequencing

We report all-atom molecular dynamics (MD) simulations of single-stranded DNA (ssDNA) translocation in 1 M KCl solution through a silicon nitride solid-state nanopore with one or two nanochannels p...

DNA Sequencing Modified Method through Effective Regulation of Its Translocation Speed in Aqueous Solution

Solid-state nanopore DNA sequencing modified method is developed. Method is based on the tunnel current investigation through the nanogap made on lateral gold electrodes in the form of nanowires or nanoribbons. The movement of DNA in aqueous solution is regulated by the potential applied to reference electrode. The potential applied to the lateral metal electrodes helps to the creation of the molecular junctions. They consist of the nucleosides passing through the pores. Taking into account that DNA moves under gravity, electrophoretic and drag forces, the analytic expression for the DNA translocation speed is calculated and analyzed. The conditions for decreasing the DNA translocation speed or increasing the nucleosides reading time are received. It is shown that one can control value of the DNA molecules bases reading time and the frequency of the bases passes by the choice of magnitude of the potential applied to reference electrode. Our results, therefore potentially suggest a realistic, inherently design-specific, high-throughput nanopore DNA sequencing device/cell as a de-novo alternative to the existing methods.

Destructing the Plasma Membrane with Activatable Vesicular DNA Nanopores.

Pore-forming proteins are an agent for attack or defense in various organisms, and its cytolytic activity has medical potential in cancer therapy. Despite recent advances in mimicking these proteins by amphipathic DNA nanopores, it remains inefficient to incorporate them into lipid bilayers. Here, we present the development of vesicular DNA nanopores that can controllably open a lipid membrane. Different from previously reported DNA nanopores that randomly insert into the planar bilayers, we design on-command fusogenic liposome-incorporated transmembrane DNA nanopores (FLIPs) that bypass the direct insertion process. By steric deshielding of fusogenic liposomal supports under low pH conditions, the embedded FLIPs are transferred and perforate lipid bilayers. We find that FLIPs depolarize the plasma membrane and thereby induce pyroptosis-like cell death. We further demonstrate the use of FLIPs to inhibit tumor growth in murine tumor models, which provides a new route to cancer nanotherapy.

A large size-selective DNA nanopore with sensing applications

Transmembrane nanostructures like ion channels and transporters perform key biological functions by controlling flow of molecules across lipid bilayers. Much work has gone into engineering artificial nanopores and applications in selective gating of molecules, label-free detection/sensing of biomolecules and DNA sequencing have shown promise. Here, we use DNA origami to create a synthetic 9 nm wide DNA nanopore, controlled by programmable, lipidated flaps and equipped with a size-selective gating system for the translocation of macromolecules. Successful assembly and insertion of the nanopore into lipid bilayers are validated by transmission electron microscopy (TEM), while selective translocation of cargo and the pore mechanosensitivity are studied using optical methods, including single-molecule, total internal reflection fluorescence (TIRF) microscopy. Size-specific cargo translocation and oligonucleotide-triggered opening of the pore are demonstrated showing that the DNA nanopore can function as a real-time detection system for external signals, offering potential for a variety of highly parallelized sensing applications.

Nanopore sequencing of the glucocerebrosidase (GBA) gene in a New Zealand Parkinson's disease cohort

IntroductionBi-allelic mutations in the gene for glucocerebrosidase (GBA) cause Gaucher disease, an autosomal recessive lysosomal storage disorder. Gaucher disease causing GBA mutations in the heterozygous state are also high risk factors for Parkinson's disease (PD). GBA analysis is challenging due to a related pseudogene and structural variations (SVs) that can occur at this locus. We have applied and refined a recently developed nanopore DNA sequencing method to analyze GBA variants in a clinically assessed New Zealand longitudinal cohort of PD. MethodWe examined amplicons encompassing the coding region of GBA (8.9 kb) from 229 PD cases and 50 healthy controls using the GridION nanopore sequencing platform, and Sanger validation. ResultsWe detected 23 variants in 21 PD cases (9.2% of patients). We detected modest PD risk variant p.N409S (rs76763715) in one case, p.E365K (rs2230288) in 12 cases, and p.T408 M (rs75548401) in seven cases, one of whom also had p.E365K. We additionally detected the possible risk variants p.R78C (rs146774384) in one case, p.D179H (rs147138516) in one case which occurred on the same haplotype as p.E365K, and one novel variant c.335C > T or p.(L335 = ), that potentially impacts splicing of GBA transcripts. Additionally, we found a higher prevalence of dementia among patients with GBA variants. ConclusionThis work confirmed the utility of nanopore sequencing as a high-throughput method to identify known and novel GBA variants, and to assign precise haplotypes. Our observations may contribute to improved understanding of the effects of variants on disease pathogenesis, and to the development of more targeted treatments.

Tailfindr: Alignment-free poly(A) length measurement for Oxford Nanopore RNA and DNA sequencing

tailfindr: Alignment-free poly(A) length measurement for Oxford Nanopore RNA and DNA sequencing

Solid-state nanopores towards single-molecule DNA sequencing.

Nanopore DNA sequencing offers a new paradigm owing to its extensive potential for long-read, high-throughput detection of nucleotide modification and direct RNA sequencing. Given the remarkable advances in protein nanopore sequencing technology, there is still a strong enthusiasm in exploring alternative nanopore-sequencing techniques, particularly those based on a solid-state nanopore using a semiconductor material. Since solid-state nanopores provide superior material robustness and large-scale integrability with on-chip electronics, they have the potential to surpass the limitations of their biological counterparts. However, there are key technical challenges to be addressed: the creation of an ultrasmall nanopore, fabrication of an ultrathin membrane, control of the ultrafast DNA speed and detection of four nucleotides. Extensive research efforts have been devoted to resolving these issues over the past two decades. In this review, we briefly introduce recent updates regarding solid-state nanopore technologies towards DNA sequencing. It can be envisioned that emerging technologies will offer a brand new future in DNA-sequencing technology.

Rapid metagenomics analysis of EMS vehicles for monitoring pathogen load using nanopore DNA sequencing.

Pathogen monitoring, detection and removal are essential to public health and outbreak management. Systems are in place for monitoring the microbial load of hospitals and public health facilities with strategies to mitigate pathogen spread. However, no such strategies are in place for ambulances, which are tasked with transporting at-risk individuals in immunocompromised states. As standard culturing techniques require a laboratory setting, and are time consuming and labour intensive, our approach was designed to be portable, inexpensive and easy to use based on the MinION third-generation sequencing platform from Oxford Nanopore Technologies. We developed a transferable sampling-to-analysis pipeline to characterize the microbial community in emergency medical service vehicles. Our approach identified over sixty-eight organisms in ambulances to the genera level, with a proportion of these being connected with health-care associated infections, such as Clostridium spp. and Staphylococcus spp. We also monitored the microbiome of different locations across three ambulances over time, and examined the dynamic community of microorganisms found in emergency medical service vehicles. Observed differences identified hot spots, which may require heightened monitoring and extensive cleaning. Through metagenomics analysis it is also possible to identify how microorganisms spread between patients and colonize an ambulance over time. The sequencing results aid in the development of practices to mitigate disease spread, while also providing a useful tool for outbreak prediction through ongoing analysis of the ambulance microbiome to identify new and emerging pathogens. Overall, this pipeline allows for the tracking and monitoring of pathogenic microorganisms of epidemiological interest, including those related to health-care associated infections.

Tailfindr: alignment-free poly(A) length measurement for Oxford Nanopore RNA and DNA sequencing.

Polyadenylation at the 3′-end is a major regulator of messenger RNA and its length is known to affect nuclear export, stability, and translation, among others. Only recently have strategies emerged that allow for genome-wide poly(A) length assessment. These methods identify genes connected to poly(A) tail measurements indirectly by short-read alignment to genetic 3′-ends. Concurrently, Oxford Nanopore Technologies (ONT) established full-length isoform-specific RNA sequencing containing the entire poly(A) tail. However, assessing poly(A) length through base-calling has so far not been possible due to the inability to resolve long homopolymeric stretches in ONT sequencing. Here we present tailfindr, an R package to estimate poly(A) tail length on ONT long-read sequencing data. tailfindr operates on unaligned, base-called data. It measures poly(A) tail length from both native RNA and DNA sequencing, which makes poly(A) tail studies by full-length cDNA approaches possible for the first time. We assess tailfindr’s performance across different poly(A) lengths, demonstrating that tailfindr is a versatile tool providing poly(A) tail estimates across a wide range of sequencing conditions.

Nanopore metagenomics enables rapid clinical diagnosis of bacterial lower respiratory infection.

The gold standard for clinical diagnosis of bacterial lower respiratory infections (LRIs) is culture, which has poor sensitivity and is too slow to guide early, targeted antimicrobial therapy. Metagenomic sequencing could identify LRI pathogens much faster than culture, but methods are needed to remove the large amount of human DNA present in these samples for this approach to be feasible. We developed a metagenomics method for bacterial LRI diagnosis that features efficient saponin-based host DNA depletion and nanopore sequencing. Our pilot method was tested on 40 samples, then optimized and tested on a further 41 samples. Our optimized method (6 h from sample to result) was 96.6% sensitive and 41.7% specific for pathogen detection compared with culture and we could accurately detect antibiotic resistance genes. After confirmatory quantitative PCR and pathobiont-specific gene analyses, specificity and sensitivity increased to 100%. Nanopore metagenomics can rapidly and accurately characterize bacterial LRIs and might contribute to a reduction in broad-spectrum antibiotic use.

Microfluidic block copolymer membrane arrays for nanopore DNA sequencing

Nanopore DNA sequencing has the potential to provide significant improvements to DNA sequencing: it may decrease cost while increasing speed and portability. Due to fundamental limits on the speed of reading DNA as it moves through a nanopore, an array of nanopores is necessary to parallelize measurements for high speeds. Additionally, a practical nanopore sequencing device would benefit from the use of block copolymer membranes to house the nanopore proteins; block copolymers are more structurally and chemically stable than phospholipids. We have previously tailored membranes composed of a block copolymer to house the nanopore protein MspA for this purpose. In this work, we extend the use of this polymer to a membrane array. We find that when switching from our previous manual system to this microfluidic system, the nanopore protein MspA exhibits variable behavior despite the use of the same block copolymer solution as before. We establish a metric for quantifying this variability and investigate its cause. We find that the cause is likely the use of volatile and water-soluble solvents in a small channel volume. Finally, we demonstrate that MspA in these block copolymer membranes is able to translocate DNA similar to MspA behavior in lipid membranes. These results illustrate the viability of polymer membranes for nanopore-based sensors while highlighting the challenges inherent in the development of a practical nanopore DNA sequencing device.

Increasing the accuracy of nanopore DNA sequencing using a time-varying cross membrane voltage.

Nanopore DNA sequencing is limited by low base calling accuracy. Improved base-calling accuracy has so far relied on specialized base-calling algorithms, different nanopores and motor enzymes, or biochemical methods to re-read DNA molecules. Two primary error modes hamper sequencing accuracy: enzyme mis-steps and sequences with indistinguishable signals. We vary the driving voltage across an MspA nanopore between 100 to 200 mV with a frequency of 200 Hz, changing how the DNA strand moves through the nanopore. As a DNA helicase moves the DNA through the nanopore in discrete steps, the variable voltage positions the DNA continuously between these steps. The resulting electronic signal can be analysed to overcome the primary error modes in base-calling. Single-passage de novo base-calling accuracy in our device increases from 62.7 ± 0.5% with a constant driving voltage to 79.3 ± 0.3% with a variable driving voltage. Our variable-voltage sequencing mode is complementary to other advances in nanopore sequencing and is amenable to incorporation into other enzyme-actuated nanopore sequencing devices.

Electrostatic Forces in Field-Perturbed Equilibria: Nanopore Analysis of Cage Complexes

Summary Molecular recognition has been widely investigated under equilibrium conditions, but little is known about such processes when perturbed by external forces. Here, we investigate the influence of electric fields on complexes formed between metallosupramolecular cages and a protein nanopore at the single-molecule level. Association rates were dominated by the applied voltage, whereas local electrostatic interactions between the cage and the nanopore more greatly influenced the dissociation kinetics. By exploiting these principles, we showed that the externally applied voltage could be used to selectively bind a specific cage from a mixture containing a large excess of other cages. Moreover, the applied voltage could also be used to drive supramolecular enantio-inversion of the chiral cages or, occasionally, the non-equilibrium capture and disassembly of cages deep within the nanopore. Similar principles might be exploited in the design of other molecular devices that operate within externally applied electric fields or biogenic transmembrane potentials.

Targeted resequencing using the MinION long read sequencing platform

MinION nanopore DNA sequencing is an emerging paradigm in portable, long read, economic sequencing technology. Using a targeted resequencing approach of the rat osteopontin (Spp1) region in the stroke‐prone spontaneously hypertensive (SHRSPGla) and Wistar Kyoto (WKYGla) rats. We aim to develop and validate a variant calling pipeline based on long reads produced by the MinION platform and to corroborate with published SHRSPGla and WKYGla promotor variants using the Rat Genome Database (RGD) (Rnor6.0).Using the Phusion high fidelity DNA polymerase, seven PCR amplifications of defined length were amplified across a 15kb region of the rat Spp1 promoter and gene from liver samples from two individual SHRSPGla and WKYGla animals. The library for MinION sequencing was prepared using Oxford Nanopore Ligation Sequencing Kit and Native Barcoding Expansion 1–12. Purified PCR amplicons were repaired and end‐prepped with dA‐tailing. Barcodes and adapters were ligated to the prepared DNA fragments before loading to the MinION flow cell for sequencing. Two Oxford Nanopore SpotON Flow cells were primed and duplicate prepared samples were each loaded onto flow cells on two separate MinION. The MinION raw data was analysed in real time during sequencing by cloud‐based EPI2ME software (Metrichor). This enabled base calling of raw data to produce DNA sequence from output measurements of current, converting raw fast5 files to base called fastq format. Reads were separated by barcode to identify reads by strain and animal. Quality control metrics for each sample were obtained from the BAM files produced by Samtools Mpileup performed on the merged MinION fastq files mapping to the Spp1 region (Chromosome 14, 6,675,281–6,690,258). A BWA/Bcftools pipeline was used to call variants (Phred>20). Variant calls from the SHRSPGla/WKYGla strains using our MinION pipeline were cross‐referenced with previously published Illumina BWA/GATK calls on RGD for this region (Rnor6.0).The MinION platform produced a mean read count of 1.49×104(± 6.21×103) per sample, with a mean mapping percentage of 49.9% (±10.1). The mean read length was 1130 bases (±132) with a mean quality of base calls of 12.83 (±0.16). Mean depth of coverage of each individual sample was 610x(±180). RGD describes 38 variants in this region of the osteopontin promotor. Across two sequencing runs, 27 of the 38 variants listed on the Rat Genome Database were called and identified several potential novel variants in the region.In conclusion, we confirm the MinION platform can be used as a targeted resequencing platform and variant calls can be made from the long reads produced, although read accuracy is dependent on read coverage.Support or Funding InformationBritish Heart Foundation and Stratified Medicine Scotland Innovation Centre is supported by the Scottish Funding Council, Scottish Enterprise and the Chief Scientist Office of ScotlandThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Structural and Functional Stability of DNA Nanopores in Biological Media.

DNA nanopores offer a unique nano-scale foothold at the membrane interface that can help advance the life sciences as biophysical research tools or gate-keepers for drug delivery. Biological applications require sufficient physiological stability and membrane activity for viable biological action. In this report, we determine essential parameters for efficient nanopore folding and membrane binding in biocompatible cell media. The parameters are identified for an archetypal DNA nanopore composed of six interwoven strands carrying cholesterol lipid anchors. Using gel electrophoresis and fluorescence spectroscopy, the nanostructures are found to assemble efficiently in cell media, such as LB and DMEM, and remain structurally stable at physiological temperatures. Furthermore, the pores’ oligomerization state is monitored using fluorescence spectroscopy and confocal microscopy. The pores remain predominately water-soluble over 24 h in all buffer systems, and were able to bind to lipid vesicles after 24 h to confirm membrane activity. However, the addition of fetal bovine serum to DMEM causes a significant reduction in nanopore activity. Serum proteins complex rapidly to the pore, most likely via ionic interactions, to reduce the effective nanopore concentration in solution. Our findings outline crucial conditions for maintaining lipidated DNA nanodevices, structurally and functionally intact in cell media, and pave the way for biological studies in the future.

Nanometre electron beam sculpting of suspended graphene and hexagonal boron nitride heterostructures

Nano-patterned and suspended graphene membranes find applications in electronic devices, filtration and nano-pore DNA sequencing. However, the fabrication of suspended graphene structures with nanoscale features is challenging. We report the direct patterning of suspended membranes consisting of a graphene layer on top of a thin layer of hexagonal boron nitride which acts as a mechanical support, using a highly focused electron beam to fabricate structures with extremely high spatial resolution within the scanning transmission electron microscope (STEM). The boron nitride support enables the fabrication of stable graphene geometries by preventing intrinsic strain in graphene membranes from distorting the patterned features. Line cuts with widths below 2 nm are reported. We also demonstrate that the extent of cutting can be monitored in situ utilising electron energy loss spectroscopy (EELS).