Ionic Current-Based Mapping of Short Sequence Motifs in Single DNA Molecules Using Solid-State Nanopores

Nanopore sensors show great potential for rapid, single-molecule determination of DNA sequence information. Here, we develop an ionic current-based method for determining the positions of short sequence motifs in double-stranded DNA molecules with solid-state nanopores. Using the DNA-methyltransferase M.TaqI and a biotinylated S-adenosyl-l-methionine cofactor analogue we create covalently attached biotin labels at 5′-TCGA-3′ sequence motifs. Monovalent streptavidin is then added to bind to the biotinylated sites giving rise to additional current blockade signals when the DNA passes through a conical quartz nanopore. We determine the relationship between translocation time and position along the DNA contour and find a minimum resolvable distance between two labeled sites of ∼200 bp. We then characterize a variety of DNA molecules by determining the positions of bound streptavidin and show that two short genomes can be simultaneously detected in a mixture. Our method provides a simple, generic single-molecule detection platform enabling DNA characterization in an electrical format suited for portable devices for potential diagnostic applications.

[1]  B. Nordén,et al.  Peptide nucleic acid (PNA): its medical and biotechnical applications and promise for the future , 2000, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[2]  Marc Pignot,et al.  Structure of the N6-adenine DNA methyltransferase M•TaqI in complex with DNA and a cofactor analog , 2001, Nature Structural Biology.

[3]  Peer Bork,et al.  Systematic identification of novel protein domain families associated with nuclear functions. , 2002, Genome research.

[4]  Jiajun Gu,et al.  PROBING SINGLE DNA MOLECULE TRANSPORT USING FABRICATED NANOPORES. , 2004, Nano letters.

[5]  C. Dekker,et al.  Translocation of double-strand DNA through a silicon oxide nanopore. , 2005, Physical review. E, Statistical, nonlinear, and soft matter physics.

[6]  Pieter C Dorrestein,et al.  A monovalent streptavidin with a single femtomolar biotin binding site , 2006, Nature Methods.

[7]  Saulius Klimasauskas,et al.  Targeted labeling of DNA by methyltransferase-directed transfer of activated groups (mTAG). , 2007, Journal of the American Chemical Society.

[8]  Marc Gershow,et al.  Recapturing and trapping single molecules with a solid-state nanopore. , 2007, Nature nanotechnology.

[9]  Pui-Yan Kwok,et al.  Rapid DNA mapping by fluorescent single molecule detection , 2006, Nucleic acids research.

[10]  Z. Siwy,et al.  Nanopore analytics: sensing of single molecules. , 2009, Chemical Society reviews.

[11]  Meni Wanunu,et al.  Nanopore based sequence specific detection of duplex DNA for genomic profiling. , 2010, Nano letters.

[12]  Sheereen Majd,et al.  Controlling the translocation of proteins through nanopores with bioinspired fluid walls , 2011, Nature nanotechnology.

[13]  E. Weinhold,et al.  Sequence-specific covalent labelling of DNA. , 2011, Biochemical Society transactions.

[14]  S. Muller,et al.  Labeling DNA for single-molecule experiments: methods of labeling internal specific sequences on double-stranded DNA. , 2011, Nanoscale.

[15]  J. Hofkens,et al.  Optical mapping of DNA: Single‐molecule‐based methods for mapping genomes , 2011, Biopolymers.

[16]  Fernando Albertorio,et al.  Origins and consequences of velocity fluctuations during DNA passage through a nanopore. , 2011, Biophysical journal.

[17]  Aleksei Aksimentiev,et al.  Slowing down DNA translocation through a nanopore in lithium chloride. , 2012, Nano letters.

[18]  D. Ly,et al.  Electronic barcoding of a viral gene at the single-molecule level. , 2012, Nano letters.

[19]  Joshua B Edel,et al.  Single molecule sensing with solid-state nanopores: novel materials, methods, and applications. , 2013, Chemical Society reviews.

[20]  D. Stein,et al.  Statistics of DNA capture by a solid-state nanopore. , 2012, Physical review letters.

[21]  A. Hall,et al.  Selective detection and quantification of modified DNA with solid-state nanopores. , 2014, Nano letters.

[22]  E. Weinhold,et al.  Sequence-specific labeling of nucleic acids and proteins with methyltransferases and cofactor analogues. , 2014, Journal of visualized experiments : JoVE.

[23]  Robert Tjian,et al.  CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells , 2015, Proceedings of the National Academy of Sciences.

[24]  Ki-Bum Kim,et al.  Identifying the Location of a Single Protein along the DNA Strand Using Solid-State Nanopores. , 2015, ACS nano.

[25]  C. Dekker,et al.  Detection of Individual Proteins Bound along DNA Using Solid-State Nanopores. , 2015, Nano letters.

[26]  Assaf Grunwald,et al.  Bacteriophage strain typing by rapid single molecule analysis , 2015, Nucleic acids research.

[27]  A. Radenović,et al.  Relevance of the Drag Force during Controlled Translocation of a DNA-Protein Complex through a Glass Nanocapillary. , 2015, Nano letters.

[28]  Nicholas A. W. Bell,et al.  Specific Protein Detection Using Designed DNA Carriers and Nanopores , 2015, Journal of the American Chemical Society.

[29]  S. Maerkl,et al.  Single Molecule Localization and Discrimination of DNA-Protein Complexes by Controlled Translocation Through Nanocapillaries. , 2016, Nano letters.

[30]  Ulrich F Keyser,et al.  Translocation frequency of double-stranded DNA through a solid-state nanopore. , 2015, Physical review. E.

[31]  Ulrich F. Keyser,et al.  Digitally encoded DNA nanostructures for multiplexed, single-molecule protein sensing with nanopores. , 2016, Nature nanotechnology.

[32]  Z. Siwy,et al.  Nanopores and Nanochannels: From Gene Sequencing to Genome Mapping. , 2016, ACS nano.

[33]  Tza-Huei Wang,et al.  Analysis of single nucleic acid molecules in micro- and nano-fluidics. , 2016, Lab on a chip.

[34]  Harold Riethman,et al.  CRISPR-CAS9 D10A nickase target-specific fluorescent labeling of double strand DNA for whole genome mapping and structural variation analysis , 2015, Nucleic acids research.

[35]  Nicholas A. W. Bell,et al.  Quantifying Nanomolar Protein Concentrations Using Designed DNA Carriers and Solid-State Nanopores , 2016, Nano letters.

[36]  Sang-Hyun Oh,et al.  Nanopore sensing at ultra-low concentrations using single-molecule dielectrophoretic trapping , 2016, Nature Communications.

[37]  E. Eisenberg,et al.  Super-Resolution Genome Mapping in Silicon Nanochannels. , 2016, ACS nano.

[38]  Nicholas A. W. Bell,et al.  Direction- and Salt-Dependent Ionic Current Signatures for DNA Sensing with Asymmetric Nanopores. , 2017, Biophysical journal.