Nanopore Based Sequence Specific Detection of Duplex DNA for Genomic Profiling

Abstract

We demonstrate a purely electrical method for single-molecule detection of specific DNA sequences, achieved by hybridizing double-stranded DNA (dsDNA) with peptide nucleic acid (PNA) probes and electrophoretically threading the DNA through sub-5 nm silicon nitride pores. Bis-PNAs were used as the tagging probes, in order to achieve high affinity and sequence-specificity. Sequence detection is performed by reading the ion current traces of individual translocating DNA molecules, which display a characteristic secondary blockade level, absent in untagged molecules. The potential for barcoding DNA is demonstrated through nanopore analysis of once-tagged and twice-tagged DNA at different locations on the same genomic fragment. Our high-throughput, long-read length method can be used to identify key sequences embedded in individual DNA molecules, without the need for amplification or fluorescent/radio labeling. This opens up a wide range of possibilities in human genomics, as well as in pathogen detection for fighting infectious diseases. KeywordsPNA; invasion; sequence detection; nanopore; DNA Numerous techniques in life sciences, biotechnology, medicine, and forensics are based on nucleic acid hybridization. The invention of nucleic acid analogs with improved hybridization affinity, hybridization rate, and/or mismatch discrimination as compared to natural nucleic acids, has significantly extended the diagnostic utilities of these applications. Peptide nucleic acids (PNAs), a prominent class of artificial nucleic acid analogs, are neutral, oligomers with peptide-like backbone onto which nucleobases are grafted in a designed sequence. Moreover, bis-PNA molecules, consisting of two PNA oligomers connected by a flexible linker, spontaneously invade double-stranded DNA (dsDNA) molecules, binding to one of the two dsDNA strands with high affinity and sequence-specificity, owing to the simultaneous formation of Watson-Crick and Hoogsteen base-pairs 1-3 . This high affinity and sequencespecificity makes bis-PNA, and other synthetic variants (e.g., pseudocomplementary PNA 2, 4 and γ-PNA 5, 6 ) extremely promising sequence-tagging candidates for analysis of individual dsDNA fragments. Single-molecule mapping methods, which detect and localize PNA/DNA hybridization on minute quantities of dsDNA can lead to cheaper and faster pathogen and mutation diagnostics platforms. Low-cost and high speed platforms are essential for effective response to emerging threats of infection and will ultimately result in more accurate treatment, as well as an overall decrease in morbidity and mortality. While a

Translocation of biomolecules through solid‐state nanopores: Theory meets experiments

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“…This involved the hybridization of ds-DNA with peptide nucleic acid probes to discriminate between untagged and tagged DNA molecules. 16 Continuous base identification was also achieved for ss-DNA translocating through biological pores. 13 Single-base resolution implies the ability to detect the different electronic signatures of the bases.…”

Artificial Nucleic Acid Probes and Their Applications in Clinical Microbiology

“…Solid-state nanopores are novel biosensors capable of detecting DNA molecules as they pass, one-by-one, through them. By fabricating single sub-5 nm pores (only slightly larger than the diameter of the dsDNA helix) in a 30 nm thick silicon-nitride membrane, and by tagging (invading) the duplex structure with bis-PNAs (Singer et al, 2010) they found that as The PD-loop design employing two bis-PNA probes with a standard molecular beacon enable the detection of target sequences in a highly sequence-specific manner as not one, but two PNAs are required to 'open-up' or expose the molecular beacon's target region.…”

Solid-State Nanopore Sensors for Nucleic Acid Analysis

“…An innovative approach involving the introduction of highly invasive peptide nucleic acid (PNA) probes was used to label target genomes with high affinity and sequence specificity, creating local bulges (P-loops) in the molecule (figure 8c). [106] Translocation of this labeled molecule resulted in secondary DNA-PNA blockade levels, effectively barcoding a target genome. While further studies are needed to determine the ultimate spatial resolution of this technique, this methodology could potentially enable the rapid, accurate, and amplification-free identification of small 5-10 kb viral genomes including hepatitis C, dengue and West Nile virus.…”