Single molecule based SNP detection using designed DNA carriers and solid-state nanopores

Abstract DNA strand displacement reaction is essential for the development of molecular computing based on DNA nanotechnology. Additional DNA strand exchange strategies with high selectivity for input will enable novel complex systems including biosensing applications. Most approaches use bulk readout methods based on fluorescent probes that complicate the monitoring of parallel computations. Herein we propose an autocatalytic strand displacement (ACSD) circuit, which is initiated by DNA breathing and accelerated by seesaw catalytic reaction. The special initiation mechanism of the ACSD circuit enables detection of single base mutations at multiple sites in the input strand with much higher sensitivity than classic toehold-mediated strand displacement. A swarm intelligence model is constructed using the ACSD circuit to mimic foraging behaviour of ants. We introduce a multiplexed nanopore sensing platform to report the output results of a parallel path selection system on the single-molecule level. The ACSD strategy and nanopore multiplexed readout method enhance the toolbox for the future development of DNA computing.

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Highly specific SNP detection using 2D graphene electronics and DNA strand displacement

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1603753113 ◽

2016 ◽

Vol 113 (26) ◽

pp. 7088-7093 ◽

Cited By ~ 56

Author(s):

Michael T. Hwang ◽

Preston B. Landon ◽

Joon Lee ◽

Duyoung Choi ◽

Alexander H. Mo ◽

...

Keyword(s):

Personalized Medicine ◽

High Specificity ◽

Practical Implementation ◽

Strand Displacement ◽

Snp Detection ◽

Single Nucleotide ◽

Specificity And Sensitivity ◽

Dna Strand Displacement ◽

Wide Range ◽

Dna Strand

Single-nucleotide polymorphisms (SNPs) in a gene sequence are markers for a variety of human diseases. Detection of SNPs with high specificity and sensitivity is essential for effective practical implementation of personalized medicine. Current DNA sequencing, including SNP detection, primarily uses enzyme-based methods or fluorophore-labeled assays that are time-consuming, need laboratory-scale settings, and are expensive. Previously reported electrical charge-based SNP detectors have insufficient specificity and accuracy, limiting their effectiveness. Here, we demonstrate the use of a DNA strand displacement-based probe on a graphene field effect transistor (FET) for high-specificity, single-nucleotide mismatch detection. The single mismatch was detected by measuring strand displacement-induced resistance (and hence current) change and Dirac point shift in a graphene FET. SNP detection in large double-helix DNA strands (e.g., 47 nt) minimize false-positive results. Our electrical sensor-based SNP detection technology, without labeling and without apparent cross-hybridization artifacts, would allow fast, sensitive, and portable SNP detection with single-nucleotide resolution. The technology will have a wide range of applications in digital and implantable biosensors and high-throughput DNA genotyping, with transformative implications for personalized medicine.

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First passage time study of DNA strand displacement

10.1101/2020.05.21.109454 ◽

2020 ◽

Author(s):

D. W. Bo Broadwater ◽

Alexander W. Cook ◽

Harold D. Kim

Keyword(s):

Single Molecule ◽

First Passage Time ◽

Resonance Energy ◽

Passage Time ◽

Single Step ◽

Strand Displacement ◽

First Passage ◽

Dna Strand Displacement ◽

Dna Strand ◽

Kinetics Of

AbstractDNA strand displacement, where a single-stranded nucleic acid invades a DNA duplex, is pervasive in genomic processes and DNA engineering applications. The kinetics of strand displacement have been studied in bulk; however, the kinetics of the underlying strand exchange were obfuscated by a slow bimolecular association step. Here, we use a novel single-molecule Fluorescence Resonance Energy Transfer (smFRET) approach termed the “fission” assay to obtain the full distribution of first passage times of unimolecular strand displacement. At a frame time of 4.4 ms, the first passage time distribution for a 14-nt displacement domain exhibited a nearly monotonic decay with little delay. Among the eight different sequences we tested, the mean displacement time was on average 35 ms and varied by up to a factor of 13. The measured displacement kinetics also varied between complementary invaders and between RNA and DNA invaders of the same base sequence except for T→U substitution. However, displacement times were largely insensitive to the monovalent salt concentration in the range of 0.25 M to 1 M. Using a one-dimensional random walk model, we infer that the single-step displacement time is in the range of ∼30 µs to ∼300 µs depending on the base identity. The framework presented here is broadly applicable to the kinetic analysis of multistep processes investigated at the single-molecule level.

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A nanopore interface for high bandwidth DNA computing

10.1101/2021.12.30.474555 ◽

2021 ◽

Author(s):

Karen Zhang ◽

Yuan-Jyue Chen ◽

Kathryn Doroschak ◽

Karin Strauss ◽

Luis Ceze ◽

...

Keyword(s):

Fluorescence Spectroscopy ◽

Single Molecule ◽

Dna Computing ◽

Direct Detection ◽

New Paradigm ◽

Single Molecule Level ◽

Oxford Nanopore ◽

Dna Strand Displacement ◽

Order Of Magnitude ◽

Dna Strand

DNA has emerged as a powerful substrate for programming information processing machines at the nanoscale. Among the DNA computing primitives used today, DNA strand displacement (DSD) is arguably the most popular, with DSD-based circuit applications ranging from disease diagnostics to molecular artificial neural networks. The outputs of DSD circuits are generally read using fluorescence spectroscopy. However, due to the spectral overlap of typical small-molecule fluorescent reporters, the number of unique outputs that can be detected in parallel is limited, requiring complex optical setups or spatial isolation of reactions to make output bandwidths scalable. Here, we present a multiplexable sequencing-free readout method that enables real-time, kinetic measurement of DSD circuit activity through highly parallel, direct detection of barcoded output strands using nanopore sensor array technology (Oxford Nanopore Technologies' MinION device). We show that engineered reporter probes can be detected and classified with high accuracy at the single-molecule level directly from raw nanopore signals using deep learning. We then demonstrate this method's utility in multiplexed detection of clinically relevant microRNA sequences. These results increase DSD output bandwidth by an order of magnitude over what is possible with fluorescence spectroscopy, laying the foundations for a new paradigm in DNA circuit readout and programmable multiplexed molecular diagnostics using portable nanopore devices.

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Cover Feature: Sub-Ensemble Monitoring of DNA Strand Displacement Using Multiparameter Single-Molecule FRET (ChemPhysChem 5/2018)

ChemPhysChem ◽

10.1002/cphc.201800146 ◽

2018 ◽

Vol 19 (5) ◽

pp. 548-548

Author(s):

Laura E. Baltierra-Jasso ◽

Michael J. Morten ◽

Steven W. Magennis

Keyword(s):

Single Molecule ◽

Strand Displacement ◽

Single Molecule Fret ◽

Dna Strand Displacement ◽

Dna Strand

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Monitoring G-quadruplex formation with DNA carriers and solid-state nanopores

10.1101/788513 ◽

2019 ◽

Author(s):

Filip Bošković ◽

Jinbo Zhu ◽

Kaikai Chen ◽

Ulrich F. Keyser

Keyword(s):

Solid State ◽

Single Molecule ◽

Drug Targets ◽

Gene Expression Regulation ◽

Single Stranded Dna ◽

Single Molecule Level ◽

G Quadruplex ◽

Human Viruses ◽

Kinetics Of ◽

Dna Carriers

ABSTRACTG-quadruplexes (Gq) are guanine-rich DNA structures formed by single-stranded DNA. They are of paramount significance to gene expression regulation, but also drug targets for cancer and human viruses. Current ensemble and single-molecule methods require fluorescent labels, which can affect Gq folding kinetics. Here we introduce, a single-molecule Gq nanopore assay (smGNA) to detect Gqs and kinetics of Gq formation. We use ~5 nm solid-state nanopores to detect various Gq structural variants attached to designed DNA carriers. Gqs can be identified by localizing their positions along designed DNA carriers establishing smGNA as a tool for Gq mapping. In addition, smGNA allows for discrimination of (un-)folded Gq structures, provides insights into single-molecule kinetics of G-quadruplex folding, and probes quadruplex-to-duplex structural transitions. smGNA can elucidate the formation of G-quadruplexes at the single-molecule level without labelling and has potential implications on the study of these structures both in single-stranded DNA and in genomic samples.

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