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Single-Molecule DNA Methylation Quantification Using Electro-optical Sensing in Solid-State
Detection of epigenetic markers, including 5-methylcytosine, is crucial due to their role in gene expression regulation. To this
end, we have developed a labeling and single-molecule quantification method for multiple unmethylated cytosine−guanine
dinucleotides (CpGs). Our method involves a single-step covalent coupling of DNA with synthetic cofactor analogues using
DNA methyltransferases (MTases) followed by molecule-by-molecule electro-optical nanopore detection and quantification with
single or multiple colors. This sensing method yields a calibrated scale to directly quantify the number of unmethylated CpGs in
the target sequences of each DNA molecule.
Meller, DOI: 10.1021/acsnano.6b04748
Light-Enhancing Plasmonic-Nanopore Biosensor for Superior Single-Molecule Detection
In this study we focused on the optical enhancing properties of plasmonic nanostructures coupled to a single-molecule nanopore sensor. To this end we fabricated, experimentally characterized, and simulated PNW–NP devices designed to improve single-molecule light detection efficiency. These devices exhibit strong fluorescence background suppression and a tenfold net enhancement in the observed fluorescence intensity resulting at an extremely bright fluorescence. These results open new directions for single-molecule electro-optical sensing.
Sensing Native Protein Solution Structures Using a Solid-state Nanopore: Unraveling the States of VEGF
Monitoring individual proteins in solution while simultaneously obtaining tertiary and quaternary structural information is challenging. In this study, translocation of the vascular endothelial growth factor (VEGF) protein through a solid-state nanopore (ssNP) produces distinct ion-current blockade amplitude levels and durations likely corresponding to monomer, dimer, and higher oligomeric states. Our study shows that careful characterization of ssNP results elucidates real-time structural information about the protein, thereby complementing classical techniques for structural analysis of proteins in solution with the added advantage of quantitative single-molecule resolution of native proteins.
Real-time visualization and sub-diffraction limit localization of nanometer-scale pore formation by dielectric breakdown
Herein, we introduce synchronous, real-time, electro-optical monitoring of nanopore formation by dielectric breakdown. Using the same principle as sub-diffraction microscopy, our nanopore localization platform based on wide-field microscopy and calcium indicators provides nanoscale sensitivity. This enables us to establish critical limitations of the fabrication process and improve its reliability. In particular, we find that under certain conditions, multiple nanopores may form and that nanopores may preferentially localize at the membrane junction, either of which potentially render nanopore sensing ineffective.
Meller DOI: 10.1039/c7nr02629c
Optically-Monitored Nanopore Fabrication Using a Focused Laser Beam
ELEGANT TITLESolid-state nanopores (ssNPs) are extremely versatile single-molecule sensors and their potential have been established in numerous biomedical applications. However, the fabrication of ssNPs remains the main bottleneck to their widespread use. Herein, we introduce a rapid and localizable ssNPs fabrication method based on feedback-controlled optical etching. We show that a focused blue laser beam irreversibly etches silicon nitride (SiNx) membranes in solution. Furthermore, photoluminescence (PL) emitted from the SiNx is used to monitor the etching process in real-time, hence permitting rate adjustment. Given the total control over the nanopore position, this easily implemented method is ideally suited for electro- optical sensing and opens up the possibility of fabricating large nanopore arrays in situ.
Exploring DNA–protein interactions on the single DNA molecule level using nanofluidic tools
In this review we describe how nanofluidic devices have become an important tool to investigate how proteins interact with DNA. We describe the advantages and challenges with using nanofluidics for this purpose and highlight many of the most important papers in this field
Noise reduction in single time frame optical DNA maps
We here introduce a computationally fast method for reducing noise in single-time-frame DNA barcodes (snap-shot optical maps). By comparing to a database of more than 3000 theoretical plasmid barcodes we show that the plasmid identification capabilities are improved by our filtered single-time-frame barcodes compared to unfiltered
Westerlund DOI: 10.1371/journal.pone.0179041
Facilitated sequence assembly using densely labeled optical DNA barcodes: A combinatorial auction approach
The output from whole genome sequencing is a set of contigs, i.e. short non-overlapping DNA sequences (sizes 1-100 kilobasepairs). Here we introduce a new method for piecing together such contigs using DNA barcodes from competitive binding experiments as scaffolds.
we utilize single-molecule tracking and super-resolution localization in order to improve the mapping accuracy and resolving power of this genome mapping technique and achieve a 15-fold increase in resolving power compared to currently practiced methods. We took advantage of a naturally occurring genetic repeat array and labeled each repeat with custom-designed Trolox conjugated fluorophores for enhanced photostability. This model system allowed us to acquire extremely long image sequences of the equally spaced fluorescent markers along DNA molecules, enabling detailed characterization of nanoconfined DNA dynamics and quantitative comparison to the Odijk theory for confined polymer chains. We present a simple method to overcome the thermal fluctuations in the nanochannels and exploit single-step photobleaching to resolve subdiffraction spaced fluorescent markers along fluctuating DNA molecules with ∼100 bp resolution. In addition, we show how time-averaging over just ∼50 frames of 40 ms enhances mapping accuracy, improves mapping P-value scores by 3 orders of magnitude compared to nonaveraged alignment, and provides a significant advantage for analyzing structural variations between DNA molecules with similar sequence composition.
We present a long-read, highly sensitive single-molecule mapping technology that generates hybrid genetic/epigenetic profiles of native chromosomal DNA. The genome-wide distribution of 5-hmC in human peripheral blood cells correlates well with 5-hmC DNA immunoprecipitation (hMeDIP) sequencing. However, the long single-molecule read-length of 100 kbp to 1 Mbp produces 5-hmC profiles across variable genomic regions that failed to show up in the sequencing data
We assessed 5hmC levels in DNA extracted from a set of colon and blood cancer samples and compared 5hmC levels with healthy controls, in a single-molecule approach.
Using our method, we observed a significantly reduced level of 5hmC in blood and colon cancers and could distinguish between colon tumor and colon tissue adjacent to the tumor based on the global levels of this molecular biomarker.
EBENSTEIN doi: 10.1186/s13148-017-0368-9
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