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

Abstract

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.

Controlling DNA Tug‐of‐War in a Dual Nanopore Device

“…As two‐pore control eliminates folding in the majority of translocation events, the technology potentially enables access to a larger portion of the data. For comparison, in conventional single‐pore electronic barcoding, performed with a 14 nm pore, the majority of events are folded and do not possess usable barcodes so that only 30% of the events can be used. For the data shown in Figure d–f, 331/368 or 90% of events are unfolded.…”

“…When DNA in the kbp size range passes through a large (>5 nm) nanopore, the probability of folding occurring during translocation is high (≈60–70% for pores in the 10–14 nm range …”

“…Solid‐state nanopores lack a translocation control mechanism that can slow‐down the DNA while ensuring a high sensing voltage to generate ample signal to noise ratio (SNR) for robust feature detection. In addition, for large diameter (>5 nm) pores that enable efficient capture of long (>10 kbp) DNA, the occurrence of folds during translocation greatly reduces the number of usable events featuring a linear correspondence between translocation time and sequence position . Recently, a new class of “two‐pore” devices has been developed that enables simultaneous capture of a single DNA molecule by two closely spaced pores .…”

The applications of nanopores in studies of proteins

“…10). Last but not least, the same group also developed a solid-state nanoporebased method to recognize specific DNA motifs [123]. Using DNA methyltransferase, they covalently attached biotin labels at 5 0 -TCGA-3 0 sequence motifs, enabling the position and number of the motifs to be estimated from the secondary ionic current drop caused by bound streptavidin.…”

Mapping shifts in nanopore signal to changes in protein and protein-DNA conformation

“…(15) More recently, MSA acted as a critical component in a scheme to detect single nucleotide polymorphism, (16) and to map the positions of short sequences in longer dsDNA. (17) Despite the growing body of applied nanopore research into streptavidin-based complexes, no studies have compared the characteristics of passage and capture of the protein itself or the protein-DNA complex under widely varying conditions (i.e. salt type/concentration, voltage magnitude, pore size, etc.)…”