scholarly journals A nanopore interface for high bandwidth DNA computing

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.

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

2017 ◽  
Vol 53 (2) ◽  
pp. 436-439 ◽  
Author(s):  
Jinglin Kong ◽  
Jinbo Zhu ◽  
Ulrich F. Keyser
Keyword(s):  
Solid State ◽  
Single Molecule ◽  
Strand Displacement ◽  
Snp Detection ◽  
Single Molecule Level ◽  
Dna Strand Displacement ◽  
Dna Strand ◽  
Dna Carriers

A novel nanopore-DNA carrier method is demonstrated for SNP detection and following DNA strand displacement kinetics at the single molecule level.

scholarly journals Multiplexed direct detection of barcoded protein reporters on a nanopore array

2019 ◽  
Author(s):  
Nicolas Cardozo ◽  
Karen Zhang ◽  
Katie Doroschak ◽  
Aerilynn Nguyen ◽  
Zoheb Siddiqui ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Direct Detection ◽  
Biological Activities ◽  
Sensor Technology ◽  
One Pot ◽  
Single Molecule Level ◽  
Dna And Rna ◽  
Oxford Nanopore ◽  
Reporter Proteins

AbstractGenetically encoded reporter proteins are a cornerstone of molecular biology. While they are widely used to measure many biological activities, the current number of uniquely addressable reporters that can be used together for one-pot multiplexed tracking is small due to overlapping detection channels such as fluorescence. To address this, we built an expanded library of orthogonally-barcoded Nanopore-addressable protein Tags Engineered as Reporters (NanoporeTERs), which can be read and demuxed by nanopore sensors at the single-molecule level. By adapting a commercially available nanopore sensor array platform typically used for real-time DNA and RNA sequencing (Oxford Nanopore Technologies’ MinION), we show direct detection of NanoporeTER expression levels from unprocessed bacterial culture with no specialized sample preparation. These results lay the foundations for a new class of reporter proteins to enable multiplexed, real-time tracking of gene expression with nascent nanopore sensor technology.

scholarly journals DNA breathing initiated strand displacement circuits for mimicking ant foraging with nanopore readout

2021 ◽  
Author(s):  
Jinbo Zhu ◽  
Jinglin Kong ◽  
Ulrich Keyser ◽  
Erkang Wang
Keyword(s):  
Single Molecule ◽  
Dna Nanotechnology ◽  
Path Selection ◽  
Strand Displacement ◽  
Initiation Mechanism ◽  
Single Molecule Level ◽  
Sensing Platform ◽  
Dna Strand Displacement ◽  
Dna Strand Exchange ◽  
Dna Strand

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.

Development of DNA computing and information processing based on DNA-strand displacement

2015 ◽  
Vol 58 (10) ◽  
pp. 1515-1523 ◽  
Author(s):  
Yafei Dong ◽  
Chen Dong ◽  
Fei Wan ◽  
Jing Yang ◽  
Cheng Zhang
Keyword(s):  
Information Processing ◽  
Dna Computing ◽  
Strand Displacement ◽  
Dna Strand Displacement ◽  
Dna Strand

scholarly journals Single molecule vs. large area design of molecular electronic devices incorporating an efficient 2-aminepyridine double anchoring group

Nanoscale ◽  
2019 ◽  
Vol 11 (34) ◽  
pp. 15871-15880 ◽  
Author(s):  
L. Herrer ◽  
A. Ismael ◽  
S. Martín ◽  
D. C. Milan ◽  
J. L. Serrano ◽  
...  
Keyword(s):  
Electrical Properties ◽  
Single Molecule ◽  
Electronic Devices ◽  
Large Area ◽  
Molecular Electronic ◽  
Single Molecule Level ◽  
Order Of Magnitude ◽  
Anchoring Group ◽  
Molecular Skeleton ◽  
Molecular Electronic Devices

The electrical properties of a bidentate molecule in both large area devices and at the single molecule level have been explored and exhibit a conductance one order of magnitude higher than that of monodentate materials with same molecular skeleton.

scholarly journals Logical Inference by DNA Strand Algebra

2016 ◽  
Vol 12 (01) ◽  
pp. 29-44
Author(s):  
Kumar S. Ray ◽  
Mandrita Mondal
Keyword(s):  
Expert System ◽  
Paradigm Shift ◽  
Dna Computing ◽  
Modus Ponens ◽  
Logical Inference ◽  
Inference Mechanism ◽  
Dna Strand Displacement ◽  
Draw Conclusion ◽  
Dna Strand ◽  
Silicon Based

Based on the concept of DNA strand displacement and DNA strand algebra we have developed a method for logical inference which is not based on silicon-based computing. Essentially, it is a paradigm shift from silicon to carbon. In this paper, we have considered the inference mechanism, viz. modus ponens, to draw conclusion from any observed fact. Thus, the present approach to logical inference based on DNA strand algebra is basically an attempt to develop expert system design in the domain of DNA computing. We have illustrated our methodology with respect to the worked out example. Our methodology is very flexible for implementation of different expert system applications.

scholarly journals Oxford Nanopore sequencing-based protocol to detect CpG methylation in human mitochondrial DNA

2021 ◽  
Author(s):  
Iacopo Bicci ◽  
Claudia Calabrese ◽  
Zoe J. Golder ◽  
Aurora Gomez-Duran ◽  
Patrick F Chinnery
Keyword(s):  
Mitochondrial Dna ◽  
Single Molecule ◽  
Bisulfite Sequencing ◽  
Nuclear Dna ◽  
Cpg Methylation ◽  
Nanopore Sequencing ◽  
Single Molecule Level ◽  
Oxford Nanopore ◽  
Genome Bisulfite Sequencing ◽  
Technical Issues

SummaryMethylation on CpG residues is one of the most important epigenetic modifications of nuclear DNA, regulating gene expression. Methylation of mitochondrial DNA (mtDNA) has been studied using whole genome bisulfite sequencing (WGBS), but recent evidence has uncovered major technical issues which introduce a potential bias during methylation quantification. Here, we validate the technical concerns with WGBS, and then develop and assess the accuracy of a protocol for variant-specific methylation identification using long-read Oxford Nanopore Sequencing. Our approach circumvents mtDNA-specific confounders, while enriching for native full-length molecules over nuclear DNA. Variant calling analysis against Illumina deep re-sequencing showed that all expected mtDNA variants can be reliably identified. Methylation calling revealed negligible mtDNA methylation levels in multiple human primary and cancer cell lines. In conclusion, our protocol enables the reliable analysis of epigenetic modifications of mtDNA at single-molecule level at single base resolution, with potential applications beyond methylation.MotivationAlthough whole genome bisulfite sequencing (WGBS) is the gold-standard approach to determine base-level CpG methylation in the nuclear genome, emerging technical issues raise questions about its reliability for evaluating mitochondrial DNA (mtDNA) methylation. Concerns include mtDNA strand asymmetry rendering the C-rich light strand disproportionately vulnerable the chemical modifications introduced with WGBS. Also, short-read sequencing can result in a co-amplification of nuclear sequences originating from ancestral mtDNA with a high nucleotide similarity. Lastly, calling mtDNA alleles with varying proportions (heteroplasmy) is complicated by the C-to-T conversion introduced by WGBS on unmethylated CpGs. Here, we propose an alternative protocol to quantify methyl-CpGs in mtDNA, at single-molecule level, using Oxford Nanopore Sequencing (ONS). By optimizing the standard ONS library preparation, we achieved selective enrichment of native mtDNA and accurate single nucleotide variant and CpG methylation calling, thus overcoming previous limitations.

scholarly journals Mechanism of RPA-Facilitated Processive DNA Unwinding by the Eukaryotic CMG Helicase

2019 ◽  
Author(s):  
Hazal B. Kose ◽  
Sherry Xie ◽  
George Cameron ◽  
Melania S. Strycharska ◽  
Hasan Yardimci
Keyword(s):  
Single Molecule ◽  
Replication Fork ◽  
Double Helix ◽  
Dna Unwinding ◽  
Helicase Activity ◽  
Replicative Helicase ◽  
Double Stranded Dna ◽  
Order Of Magnitude ◽  
Dna Strand ◽  
Dna Double Helix

AbstractThe DNA double helix is unwound by the Cdc45/Mcm2-7/GINS (CMG) complex at the eukaryotic replication fork. While isolated CMG unwinds duplex DNA very slowly, its fork unwinding rate is stimulated by an order of magnitude by single-stranded DNA binding protein, RPA. However, the molecular mechanism by which RPA enhances CMG helicase activity remained elusive. Here, we demonstrate that engagement of CMG with parental double-stranded DNA (dsDNA) at the replication fork impairs its helicase activity, explaining the slow DNA unwinding by isolated CMG. Using single-molecule and ensemble biochemistry, we show that binding of RPA to the excluded DNA strand prevents duplex engagement by the helicase and speeds up CMG-mediated DNA unwinding. When stalled due to dsDNA interaction, DNA rezipping-induced helicase backtracking re-establishes productive helicase-fork engagement underscoring the significance of plasticity in helicase action. Together, our results elucidate the dynamics of CMG at the replication fork and reveal how other replisome components can mediate proper DNA engagement by the replicative helicase to achieve efficient fork progression.

scholarly journals Dark noise and retinal degeneration from D190N-rhodopsin

2020 ◽  
Vol 117 (37) ◽  
pp. 23033-23043 ◽  
Author(s):  
Daniel Silverman ◽  
Zuying Chai ◽  
Wendy W. S. Yue ◽  
Sravani Keerthi Ramisetty ◽  
Sowmya Bekshe Lokappa ◽  
...  
Keyword(s):  
Retinal Degeneration ◽  
Single Molecule ◽  
Protein Misfolding ◽  
Outer Segment ◽  
Segment Length ◽  
Single Molecule Level ◽  
Dark Noise ◽  
Order Of Magnitude ◽  
Human Mutation ◽  
Unclear Etiology

Numerous rhodopsin mutations have been implicated in night blindness and retinal degeneration, often with unclear etiology. D190N-rhodopsin (D190N-Rho) is a well-known inherited human mutation causing retinitis pigmentosa. Both higher-than-normal spontaneous-isomerization activity and misfolding/mistargeting of the mutant protein have been proposed as causes of the disease, but neither explanation has been thoroughly examined. We replaced wild-type rhodopsin (WT-Rho) in RhoD190N/WT mouse rods with a largely “functionally silenced” rhodopsin mutant to isolate electrical responses triggered by D190N-Rho activity, and found that D190N-Rho at the single-molecule level indeed isomerizes more frequently than WT-Rho by over an order of magnitude. Importantly, however, this higher molecular dark activity does not translate into an overall higher cellular dark noise, owing to diminished D190N-Rho content in the rod outer segment. Separately, we found that much of the degeneration and shortened outer-segment length of RhoD190N/WT mouse rods was not averted by ablating rod transducin in phototransduction—also consistent with D190N-Rho’s higher isomerization activity not being the primary cause of disease. Instead, the low pigment content, shortened outer-segment length, and a moderate unfolded protein response implicate protein misfolding as the major pathogenic problem. Finally, D190N-Rho also provided some insight into the mechanism of spontaneous pigment excitation.

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