Sequence-Specific Recognition of MicroRNAs and Other Short Nucleic Acids with Solid-State Nanopores

Zahid, Osama K.; Wang, Fanny; Růžička, Jan; Taylor, Ethan Will; Hall, Adam R.

doi:10.1021/acs.nanolett.6b00001

Cited by 84 publications

(77 citation statements)

References 31 publications

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“…Protein and solid-state nanopores (Figure 1A) are the basis for single-molecule measurements of a variety of analytes including ions, 1,2 single-stranded RNA and DNA, 3–10 double-stranded DNA, 11,12 proteins, 13–19 synthetic polymers, 20–23 and metallic nanoparticles. 24,25 The method is conceptually simple.…”

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confidence: 99%

MOSAIC: A Modular Single-Molecule Analysis Interface for Decoding Multistate Nanopore Data

et al. 2016

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Biological and solid-state nanometer-scale pores are the basis for numerous emerging analytical technologies for use in precision medicine. We developed Modular Single-Molecule Analysis Interface (MOSAIC), an open source analysis software that improves the accuracy and throughput of nanopore-based measurements. Two key algorithms are implemented: ADEPT, which uses a physical model of the nanopore system to characterize short-lived events that do not reach their steady-state current, and CUSUM+, a version of the cumulative sum statistical method optimized for longer events that do. We show that ADEPT detects previously unreported conductance states that occur as double-stranded DNA translocates through a 2.4 nm solid-state nanopore and reveals new interactions between short single-stranded DNA and the vestibule of a biological pore. These findings demonstrate the utility of MOSAIC and the ADEPT algorithm, and offer a new tool that can improve the analysis of nanopore-based measurements.

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confidence: 99%

MOSAIC: A Modular Single-Molecule Analysis Interface for Decoding Multistate Nanopore Data

et al. 2016

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“…For the appropriate combinations of base modification and enzymes, we observed exponential voltage-dependent event rates (Fig. 1c), characteristic of the assay 28,34 . Provided with the same total DNA concentrations (250 nM), the nearly identical event rate trends for both cases further indicated not only the similarity of the yields for the two labeling protocols, but also the reproducibility of the assay.…”

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confidence: 84%

Solid-State Nanopore Analysis of Diverse DNA Base Modifications Using a Modular Enzymatic Labeling Process

et al. 2017

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Many regulated epigenetic elements and base lesions found in genomic DNA can both directly impact gene expression and play a role in disease processes. However, due to their non-canonical nature, they are challenging to assess with conventional technologies. Here, we present a new approach for the targeted detection of diverse modified bases in DNA. We first use enzymatic components of the DNA base excision repair pathway to install an individual affinity label at each location of a selected modified base with high yield. We then probe the resulting material with a solid-state nanopore assay capable of discriminating labeled DNA from unlabeled. The technique features exceptional modularity via selection of targeting enzymes, which we establish through the detection of four DNA base elements: uracil, 8-oxoguanine, T:G mismatch, and the methyladenine analog 1,N6-ethenoadenine. Our results demonstrate the potential for quantitative nanopore assessment of a broad range of base modifications.

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“…In the table we also present data for prokaryotic ribosomal RNA (rRNA) capture, which exhibits a similar efficiency (see Supplementary Information, Section 6). Given the linear relationship between capture and concentration in solid-state nanopores 34–36 , extrapolation leads to sub-minute loading timescales for a 10 pg DNA sample (in a 1 µl volume) . For comparison, conventional magnetic bead loading of 10 kbp SMRTbell samples requires 1.5 ng of DNA and an hour loading time for ~30–35% loading efficiency, which corresponds to loading rates that are 5 orders of magnitude slower than our NZMWs.…”

Section: Voltage-driven Dna Packing Into Nzmwsmentioning

confidence: 99%

Length-independent DNA packing into nanopore zero-mode waveguides for low-input DNA sequencing

et al. 2017

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Compared to conventional methods, single molecule, real-time (SMRT) DNA sequencing exhibits longer read lengths than conventional methods, less GC per cent bias, and the ability to read DNA base modifications. However, reading DNA sequence from sub-ng quantities is impractical due to inefficient delivery of DNA molecules into the confines of zero-mode waveguides, zeptolitre optical cavities in which DNA sequencing proceeds. Here we show that the efficiency of voltage-induced DNA loading into waveguides equipped with nanopores at their floors is five orders of magnitude greater than existing methods. In addition, we find that DNA loading is nearly length-independent, unlike diffusive loading, which is biased towards shorter fragments. We demonstrate here loading and proof-of-principle four-colour sequence readout of a polymerase-bound 20,000 bp long DNA template within seconds from a sub-ng input quantity, a step towards low-input DNA sequencing and mammalian epigenomic mapping of native DNA samples.

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Sequence-Specific Recognition of MicroRNAs and Other Short Nucleic Acids with Solid-State Nanopores

Cited by 84 publications

References 31 publications

MOSAIC: A Modular Single-Molecule Analysis Interface for Decoding Multistate Nanopore Data

MOSAIC: A Modular Single-Molecule Analysis Interface for Decoding Multistate Nanopore Data

Solid-State Nanopore Analysis of Diverse DNA Base Modifications Using a Modular Enzymatic Labeling Process

Length-independent DNA packing into nanopore zero-mode waveguides for low-input DNA sequencing

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