DNA bridges: A novel platform for single-molecule sequencing and other DNA-protein interaction applications

DNA molecular combing is a technique that stretches thousands of long individual DNA molecules (up to 10 Mbp) into a parallel configuration on surface. It has previously been proposed to sequence these molecules by synthesis. However, this approach poses two critical challenges: 1-Combed DNA molecules are overstretched and therefore a nonoptimal substrate for polymerase extension. 2-The combing surface sterically impedes full enzymatic access to the DNA backbone. Here, we introduce a novel approach that attaches thousands of molecules to a removable surface, with a tunable stretching factor. Next, we dissolve portions of the surface, leaving the DNA molecules suspended as ‘bridges’. We demonstrate that the suspended molecules are enzymatically accessible, and we have used an enzyme to incorporate labeled nucleotides, as predicted by the specific molecular sequence. Our results suggest that this novel platform is a promising candidate to achieve high-throughput sequencing of Mbp-long molecules, which could have additional genomic applications, such as the study of other protein-DNA interactions.

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Shining a Spotlight on DNA: Single-Molecule Methods to Visualise DNA

Molecules ◽

10.3390/molecules24030491 ◽

2019 ◽

Vol 24 (3) ◽

pp. 491 ◽

Cited By ~ 8

Author(s):

Gurleen Kaur ◽

Jacob Lewis ◽

Antoine van Oijen

Keyword(s):

Mechanical Properties ◽

Nucleic Acids ◽

Small Molecules ◽

Single Molecule ◽

Single Molecules ◽

Dna Molecules ◽

Dna Interactions ◽

Biochemical Reactions ◽

Imaging Methods ◽

Protein Dna Interactions

The ability to watch single molecules of DNA has revolutionised how we study biological transactions concerning nucleic acids. Many strategies have been developed to manipulate DNA molecules to investigate mechanical properties, dynamics and protein–DNA interactions. Imaging methods using small molecules and protein-based probes to visualise DNA have propelled our understanding of complex biochemical reactions involving DNA. This review focuses on summarising some of the methodological developments made to visualise individual DNA molecules and discusses how these probes have been used in single-molecule biophysical assays.

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Oriented Soft DNA Curtains for Single Molecule Imaging

10.1101/2020.06.15.151662 ◽

2020 ◽

Author(s):

Aurimas Kopūstas ◽

Šarūnė Ivanovaitė ◽

Tomas Rakickas ◽

Ernesta Pocevičiūtė ◽

Justė Paksaitė ◽

...

Keyword(s):

Single Molecule ◽

Directed Assembly ◽

Dna Molecules ◽

Reaction Conditions ◽

Interacting Protein ◽

Glass Coverslip ◽

Dna Labeling ◽

Protein Dna Interactions ◽

Biophysical Studies ◽

New Strategies

AbstractOver the past twenty years, single-molecule methods have become extremely important for biophysical studies. These methods, in combination with new nanotechnological platforms, can significantly facilitate experimental design and enable faster data acquisition. A nanotechnological platform, which utilizes flow-stretch of immobilized DNA molecules, called DNA Curtains, is one of the best examples of such combinations. Here, we employed new strategies to fabricate a flow-stretch assay of stably immobilized and oriented DNA molecules using protein template-directed assembly. In our assay a protein template patterned on a glass coverslip served for directional assembly of biotinylated DNA molecules. In these arrays, DNA molecules were oriented to one another and maintained extended either by single- or both-ends immobilization to the protein templates. For oriented both-end DNA immobilization we employed heterologous DNA labeling and protein template coverage with the anti-digoxigenin antibody. In contrast to the single-end, both-ends immobilization does not require constant buffer flow for keeping DNAs in an extended configuration, allowing us to study protein-DNA interactions at more controllable reaction conditions. Additionally, we increased immobilization stability of the biotinylated DNA molecules using protein templates fabricated from traptavidin. Finally, we demonstrated that double-tethered Soft DNA Curtains can be used in nucleic acid-interacting protein (e.g. CRISPR-Cas9) binding assay that monitors binding location and position of individual fluorescently labeled proteins on DNA.

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Single bacterial resolvases first exploit, then constrain intrinsic dynamics of the Holliday junction to direct recombination

Nucleic Acids Research ◽

10.1093/nar/gkab096 ◽

2021 ◽

Author(s):

Sujay Ray ◽

Nibedita Pal ◽

Nils G Walter

Keyword(s):

Cluster Analysis ◽

Homologous Recombination ◽

Single Molecule ◽

Holliday Junction ◽

Energetic Cost ◽

Genomic Integrity ◽

Dna Molecules ◽

Single Molecule Fluorescence ◽

And Cluster Analysis ◽

Fluorescence Observation

Abstract Homologous recombination forms and resolves an entangled DNA Holliday Junction (HJ) crucial for achieving genetic reshuffling and genome repair. To maintain genomic integrity, specialized resolvase enzymes cleave the entangled DNA into two discrete DNA molecules. However, it is unclear how two similar stacking isomers are distinguished, and how a cognate sequence is found and recognized to achieve accurate recombination. We here use single-molecule fluorescence observation and cluster analysis to examine how prototypic bacterial resolvase RuvC singles out two of the four HJ strands and achieves sequence-specific cleavage. We find that RuvC first exploits, then constrains the dynamics of intrinsic HJ isomer exchange at a sampled branch position to direct cleavage toward the catalytically competent HJ conformation and sequence, thus controlling recombination output at minimal energetic cost. Our model of rapid DNA scanning followed by ‘snap-locking’ of a cognate sequence is strikingly consistent with the conformational proofreading of other DNA-modifying enzymes.

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Single-molecule spectromicroscopy: a route towards sub-wavelength refractometry

Faraday Discussions ◽

10.1039/c5fd00086f ◽

2015 ◽

Vol 184 ◽

pp. 263-274 ◽

Cited By ~ 15

Author(s):

T. A. Anikushina ◽

M. G. Gladush ◽

A. A. Gorshelev ◽

A. V. Naumov

Keyword(s):

Refractive Index ◽

Single Molecule ◽

Low Temperatures ◽

Radiative Lifetime ◽

Spatially Resolved ◽

Local Fluctuations ◽

Novel Approach ◽

Spectral Linewidth ◽

Spatial Fluctuations ◽

Sub Wavelength

We suggest a novel approach for spatially resolved probing of local fluctuations of the refractive index n in solids by means of single-molecule (SM) spectroscopy. It is based on the dependence T1(n) of the effective radiative lifetime T1 of dye centres in solids on n due to the local-field effects. Detection of SM zero-phonon lines at low temperatures gives the values of the SM natural spectral linewidth (which is inversely proportional to T1) and makes it possible to reveal the distribution of the local n values in solids. Here we demonstrate this possibility on the example of amorphous polyethylene and polycrystalline naphthalene doped with terrylene. In particular, we show that the obtained distributions of lifetime limited spectral linewidths of terrylene molecules embedded into these matrices are due to the spatial fluctuations of the refractive index local values.

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Physical manipulation of single-molecule DNA using microbead and its application to analysis of DNA–protein interaction

Journal of Magnetism and Magnetic Materials ◽

10.1016/j.jmmm.2008.11.018 ◽

2009 ◽

Vol 321 (7) ◽

pp. 655-658

Author(s):

Hirofumi Kurita ◽

Hachiro Yasuda ◽

Kazunori Takashima ◽

Shinji Katsura ◽

Akira Mizuno

Keyword(s):

Single Molecule ◽

Protein Interaction ◽

Dna Protein Interaction ◽

Physical Manipulation

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Genome-Wide Identification of 5-Methylcytosine Sites in Bacterial Genomes By High-Throughput Sequencing of MspJI Restriction Fragments

10.1101/2021.02.10.430591 ◽

2021 ◽

Author(s):

Brian P. Anton ◽

Alexey Fomenkov ◽

Victoria Wu ◽

Richard J. Roberts

Keyword(s):

Single Molecule ◽

Dna Sequences ◽

High Throughput Sequencing ◽

Cost Effective ◽

Restriction Enzymes ◽

Specific Sequence ◽

Genome Wide ◽

Cost Effective Alternative ◽

Simple Column ◽

Sequencing Platforms

ABSTRACTSingle-molecule Real-Time (SMRT) sequencing can easily identify sites of N6-methyladenine and N4-methylcytosine within DNA sequences, but similar identification of 5-methylcytosine sites is not as straightforward. In prokaryotic DNA, methylation typically occurs within specific sequence contexts, or motifs, that are a property of the methyltransferases that “write” these epigenetic marks. We present here a straightforward, cost-effective alternative to both SMRT and bisulfite sequencing for the determination of prokaryotic 5-methylcytosine methylation motifs. The method, called MFRE-Seq, relies on excision and isolation of fully methylated fragments of predictable size using MspJI-Family Restriction Enzymes (MFREs), which depend on the presence of 5-methylcytosine for cleavage. We demonstrate that MFRE-Seq is compatible with both Illumina and Ion Torrent sequencing platforms and requires only a digestion step and simple column purification of size-selected digest fragments prior to standard library preparation procedures. We applied MFRE-Seq to numerous bacterial and archaeal genomic DNA preparations and successfully confirmed known motifs and identified novel ones. This method should be a useful complement to existing methodologies for studying prokaryotic methylomes and characterizing the contributing methyltransferases.

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DISMIR: a deep learning-based cancer-detection method by integrating DNA sequence and methylation information of individual cell-free DNA reads

10.1101/2021.01.12.426440 ◽

2021 ◽

Author(s):

Jiaqi Li ◽

Lei Wei ◽

Xianglin Zhang ◽

Wei Zhang ◽

Haochen Wang ◽

...

Keyword(s):

Deep Learning ◽

Dna Sequence ◽

Cancer Detection ◽

High Throughput Sequencing ◽

Detection Method ◽

Low Cost ◽

Sequencing Data ◽

Cell Free Dna ◽

Free Dna ◽

Novel Approach

ABSTRACTDetecting cancer signals in cell-free DNA (cfDNA) high-throughput sequencing data is emerging as a novel non-invasive cancer detection method. Due to the high cost of sequencing, it is crucial to make robust and precise prediction with low-depth cfDNA sequencing data. Here we propose a novel approach named DISMIR, which can provide ultrasensitive and robust cancer detection by integrating DNA sequence and methylation information in plasma cfDNA whole genome bisulfite sequencing (WGBS) data. DISMIR introduces a new feature termed as “switching region” to define cancer-specific differentially methylated regions, which can enrich the cancer-related signal at read-resolution. DISMIR applies a deep learning model to predict the source of every single read based on its DNA sequence and methylation state, and then predicts the risk that the plasma donor is suffering from cancer. DISMIR exhibited high accuracy and robustness on hepatocellular carcinoma detection by plasma cfDNA WGBS data even at ultra-low sequencing depths. Analysis showed that DISMIR tends to be insensitive to alterations of single CpG sites’ methylation states, which suggests DISMIR could resist to technical noise of WGBS. All these results showed DISMIR with the potential to be a precise and robust method for low-cost early cancer detection.

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Genome-wide identification of 5-methylcytosine sites in bacterial genomes by high-throughput sequencing of MspJI restriction fragments

PLoS ONE ◽

10.1371/journal.pone.0247541 ◽

2021 ◽

Vol 16 (5) ◽

pp. e0247541

Author(s):

Brian P. Anton ◽

Alexey Fomenkov ◽

Victoria Wu ◽

Richard J. Roberts

Keyword(s):

Single Molecule ◽

Dna Sequences ◽

High Throughput Sequencing ◽

Cost Effective ◽

Restriction Enzymes ◽

Specific Sequence ◽

Genome Wide ◽

Cost Effective Alternative ◽

Simple Column ◽

Sequencing Platforms

Single-molecule Real-Time (SMRT) sequencing can easily identify sites of N6-methyladenine and N4-methylcytosine within DNA sequences, but similar identification of 5-methylcytosine sites is not as straightforward. In prokaryotic DNA, methylation typically occurs within specific sequence contexts, or motifs, that are a property of the methyltransferases that “write” these epigenetic marks. We present here a straightforward, cost-effective alternative to both SMRT and bisulfite sequencing for the determination of prokaryotic 5-methylcytosine methylation motifs. The method, called MFRE-Seq, relies on excision and isolation of fully methylated fragments of predictable size using MspJI-Family Restriction Enzymes (MFREs), which depend on the presence of 5-methylcytosine for cleavage. We demonstrate that MFRE-Seq is compatible with both Illumina and Ion Torrent sequencing platforms and requires only a digestion step and simple column purification of size-selected digest fragments prior to standard library preparation procedures. We applied MFRE-Seq to numerous bacterial and archaeal genomic DNA preparations and successfully confirmed known motifs and identified novel ones. This method should be a useful complement to existing methodologies for studying prokaryotic methylomes and characterizing the contributing methyltransferases.

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Novel Approach to Study the Diversity of Soil Microbial Communities in Qatar

University of the Future: Re-Imagining Research and Higher Education ◽

10.29117/quarfe.2020.0025 ◽

2020 ◽

Author(s):

Jane Oja ◽

Sakeenah Adenan ◽

Abdel-Fattah Talaat ◽

Juha Alatalo

Keyword(s):

Microbial Communities ◽

High Throughput ◽

Mycorrhizal Fungi ◽

High Throughput Sequencing ◽

Soil Microbial Communities ◽

Arbuscular Mycorrhizal ◽

Soil Microbial ◽

Fungal Abundance ◽

Novel Approach ◽

Soil Climate

A broad diversity of microorganisms can be found in soil, where they are essential for nutrient cycling and energy transfer. Recent high-throughput sequencing methods have greatly advanced our knowledge about how soil, climate and vegetation variables structure the composition of microbial communities in many world regions. However, we are lacking information from several regions in the world, e.g. Middle-East. We have collected soil from 19 different habitat types for studying the diversity and composition of soil microbial communities (both fungi and bacteria) in Qatar and determining which edaphic parameters exert the strongest influences on these communities. Preliminary results indicate that in overall bacteria are more abundant in soil than fungi and few sites have notably higher abundance of these microbes. In addition, we have detected some soil patameters, which tend to have reduced the overall fungal abundance and enhanced the presence of arbuscular mycorrhizal fungi and N-fixing bacteria. More detailed information on the diversity and composition of soil microbial communities is expected from the high-throughput sequenced data.

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HP1 proteins compact DNA into mechanically and positionally stable phase separated domains

eLife ◽

10.7554/elife.64563 ◽

2021 ◽

Vol 10 ◽

Author(s):

Madeline M Keenen ◽

David Brown ◽

Lucy D Brennan ◽

Roman Renger ◽

Harrison Khoo ◽

...

Keyword(s):

Phase Separation ◽

Single Molecule ◽

Genome Organization ◽

Stable Phase ◽

Dna Molecules ◽

Weakly Bound ◽

Dna Compaction ◽

Polymer Properties ◽

Molecular Scale ◽

Hp1 Proteins

In mammals, HP1-mediated heterochromatin forms positionally and mechanically stable genomic domains even though the component HP1 paralogs, HP1α, HP1β, and HP1γ, display rapid on-off dynamics. Here, we investigate whether phase-separation by HP1 proteins can explain these biological observations. Using bulk and single-molecule methods, we show that, within phase-separated HP1α-DNA condensates, HP1α acts as a dynamic liquid, while compacted DNA molecules are constrained in local territories. These condensates are resistant to large forces yet can be readily dissolved by HP1β. Finally, we find that differences in each HP1 paralog’s DNA compaction and phase-separation properties arise from their respective disordered regions. Our findings suggest a generalizable model for genome organization in which a pool of weakly bound proteins collectively capitalize on the polymer properties of DNA to produce self-organizing domains that are simultaneously resistant to large forces at the mesoscale and susceptible to competition at the molecular scale.

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