A Horizontal Magnetic Tweezers for Studying Single DNA Molecules and DNA-Binding Proteins

We report data from single molecule studies on the interaction between single DNA molecules and core histones using custom-designed horizontal magnetic tweezers. The DNA-core histone complexes were formed using λ-DNA tethers, core histones, and NAP1 and were exposed to forces ranging from ~2 pN to ~74 pN. During the assembly events, we observed the length of the DNA decrease in approximate integer multiples of ~50 nm, suggesting the binding of the histone octamers to the DNA tether. During the mechanically induced disassembly events, we observed disruption lengths in approximate integer multiples of ~50 nm, suggesting the unbinding of one or more octamers from the DNA tether. We also observed histone octamer unbinding events at forces as low as ~2 pN. Our horizontal magnetic tweezers yielded high-resolution, low-noise data on force-mediated DNA-core histone assembly and disassembly processes.

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Somatic mutation landscapes at single-molecule resolution

10.21203/rs.3.pex-1298/v1 ◽

2021 ◽

Author(s):

Stefanie V. Lensing ◽

Peter Ellis ◽

Federico Abascal ◽

Iñigo Martincorena ◽

Robert J. Osborne

Keyword(s):

Single Molecule ◽

Single Cells ◽

Error Rates ◽

Dna Molecules ◽

Base Pairs ◽

Single Dna Molecules ◽

Sequencing Library ◽

Somatic Mutagenesis ◽

Qpcr Quantification ◽

Duplex Sequencing

Abstract Somatic mutations drive cancer development and may contribute to ageing and other diseases. Yet, the difficulty of detecting mutations present only in single cells or small clones has limited our knowledge of somatic mutagenesis to a minority of tissues. To overcome these limitations, we introduce nanorate sequencing (NanoSeq), a new duplex sequencing protocol with error rates <5 errors per billion base pairs in single DNA molecules from cell populations. The version of the protocol described here uses clean genome fragmentation with a restriction enzyme to prevent end-repair-associated errors and ddBTPs/dATPs during A-tailing to prevent nick extension. Both changes reduce the error rate of standard duplex sequencing protocols by preventing the fixation of DNA damage into both strands of DNA molecules during library preparation. We also use qPCR quantification of the library prior to amplification to optimise the complexity of the sequencing library given the desired sequencing coverage, maximising duplex coverage. The sample preparation protocol takes between 1 and 2 days, depending on the number of samples processed. The bioinformatic protocol is described in:https://github.com/cancerit/NanoSeqhttps://github.com/fa8sanger/NanoSeq_Paper_Code

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Single-Molecule Studies of DNA, Visualization and Manipulation of Individual DNA Molecules with Fluorescence Microscopy and Optical Tweezers

10.1007/springerreference_78587 ◽

2012 ◽

Keyword(s):

Fluorescence Microscopy ◽

Optical Tweezers ◽

Single Molecule ◽

Dna Molecules ◽

Single Molecule Studies

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Single-molecule DNA visualization using AT-specific red and non-specific green DNA-binding fluorescent proteins

The Analyst ◽

10.1039/c8an01426d ◽

2019 ◽

Vol 144 (3) ◽

pp. 921-927 ◽

Cited By ~ 4

Author(s):

Jihyun Park ◽

Seonghyun Lee ◽

Nabin Won ◽

Eunji Shin ◽

Soo-Hyun Kim ◽

...

Keyword(s):

Dna Binding ◽

Single Molecule ◽

Fluorescent Proteins ◽

Physical Map ◽

Dna Molecules ◽

Single Dna Molecules

Two-color DNA physical map for efficient identification of single DNA molecules.

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Disturbance-free rapid solution exchange for magnetic tweezers single-molecule studies

Nucleic Acids Research ◽

10.1093/nar/gkv554 ◽

2015 ◽

Vol 43 (17) ◽

pp. e113-e113 ◽

Cited By ~ 19

Author(s):

Shimin Le ◽

Mingxi Yao ◽

Jin Chen ◽

Artem K. Efremov ◽

Sara Azimi ◽

...

Keyword(s):

Single Molecule ◽

Magnetic Tweezers ◽

Solution Exchange ◽

Single Molecule Studies

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Counting Single DNA Molecules in a Capillary with Radial Focusing

Australian Journal of Chemistry ◽

10.1071/ch02192 ◽

2003 ◽

Vol 56 (3) ◽

pp. 149 ◽

Cited By ~ 18

Author(s):

Jinjian Zheng ◽

Edward S. Yeung

Keyword(s):

Single Molecule ◽

Detection Efficiency ◽

Signal To Noise Ratio ◽

Sampling Rate ◽

Ccd Camera ◽

Counting Efficiency ◽

Spherical Particles ◽

Dna Molecules ◽

Single Dna Molecules ◽

Relative Standard

For single-molecule detection, usually a small detection volume of 10 pL or less is used to improve the signal-to-noise ratio. Detection of every molecule in a sample requires that the sample be driven through a well-defined volume to facilitate laser excitation. We report a novel approach to count single DNA molecules with nearly 100% efficiency. By applying an electric field across a 40 cm long, 75 × 75 µm2 square capillary together with hydrodynamic flow from cathode to anode, we were able to concentrate more than 95% of DNA molecules into a 10 µm region at the centre of the capillary. The YOYO-1 labelled λ-DNA molecules were imaged with an intensified CCD camera. We found that the single DNA molecule detection efficiency in a 10–17 M solution was 114 ± 21%. The mobility of the DNA molecules after radial focusing was relatively constant, with relative standard deviations ranging from 0.8% to 1.4%. This allowed us to match the sampling rate to the length of the detection window to maximize counting efficiency. Analysis of a 40.2 nL injected plug of 2 × 10–14 M λ-DNA gave a result of 492 ± 73 molecules, which agreed well with the estimated value of 484. This method should be generally useful for counting deformable molecules or non-spherical particles at extremely low concentrations.

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Denaturation transition of stretched DNA

Biochemical Society Transactions ◽

10.1042/bst20120298 ◽

2013 ◽

Vol 41 (2) ◽

pp. 639-645 ◽

Cited By ~ 4

Author(s):

Andreas Hanke

Keyword(s):

Nucleic Acids ◽

Single Molecule ◽

Force Spectroscopy ◽

Magnetic Tweezers ◽

Present Review ◽

Dna Molecules ◽

Force Measurements ◽

Force Extension ◽

Atomic Force Spectroscopy ◽

Atomic Force

In the last two decades, single-molecule force measurements using optical and magnetic tweezers and atomic force spectroscopy have dramatically expanded our knowledge of nucleic acids and proteins. These techniques characterize the force on a biomolecule required to produce a given molecular extension. When stretching long DNA molecules, the observed force–extension relationship exhibits a characteristic plateau at approximately 65 pN where the DNA may be extended to almost twice its B-DNA length with almost no increase in force. In the present review, I describe this transition in terms of the Poland–Scheraga model and summarize recent related studies.

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Real time observation of chaperone-modulated talin mechanics under single molecule resolution

10.1101/2021.04.27.441571 ◽

2021 ◽

Author(s):

Souradeep Banerjee ◽

Deep Chaudhuri ◽

Soham Chakraborty ◽

Shubhasis Haldar

Keyword(s):

Focal Adhesion ◽

Single Molecule ◽

Mechanical Stability ◽

Energy Landscape ◽

Magnetic Tweezers ◽

Downstream Signaling ◽

Cellular Processes ◽

Single Molecule Studies ◽

Time Observation

AbstractRecent single molecule studies have recognized talin as a mechanosensitive hub in focal adhesion, where its function is strongly regulated by mechanical force. For instance, at low force (less than 5pN), folded talin binds RIAM for integrin activation; whereas at high force (more than 5pN), it unfolds to activate vinculin binding for focal adhesion stabilization. Being a cytoplasmic large protein, talin must interact with various chaperones, however the role of chaperones on talin mechanics is unknown.To address this question, we investigated the force response of a mechanically stable talin domain, with a set of well-known holdase and foldase chaperones, using a single molecule magnetic tweezers technology. Our findings demonstrate a novel mechanical role of chaperones. We found holdase chaperones reduce the mechanical stability of the protein to ~6 pN, while the foldase chaperone increases it up to ~15 pN. The alteration in mechanical stability ascribes to the underlying molecular mechanism where the chaperones directly reshape the energy landscape of talin. For example, unfoldase chaperone (DnaK) decreases the unfolding barrier height from 26.8 to 21.69 kBT and increases the refolding barrier from 3.49 to 11.31 kBT. In contrast, foldase chaperone (DsbA) increases the unfolding barrier to 33.46 kBT and decreases the refolding barrier to 0.44 kBT. The quantitative mapping of the chaperone-induced free energy landscape of talin directly shows that chaperones could perturb the focal adhesion dynamics, which in turn can influence downstream signaling cascades in diverse cellular processes.

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Real-time detection of condensin-driven DNA compaction reveals a multistep binding mechanism

10.1101/149138 ◽

2017 ◽

Cited By ~ 3

Author(s):

Jorine M. Eeftens ◽

Shveta Bisht ◽

Jacob Kerssemakers ◽

Christian H. Haering ◽

Cees Dekker

Keyword(s):

Real Time ◽

Single Molecule ◽

Electrostatic Interactions ◽

Atp Hydrolysis ◽

Protein Complexes ◽

Magnetic Tweezers ◽

Condensed State ◽

Binding Modes ◽

Dna Molecules ◽

Dna Compaction

ABSTRACTCondensin, a conserved member of the SMC protein family of ring-shaped multi-subunit protein complexes, is essential for structuring and compacting chromosomes. Despite its key role, its molecular mechanism has remained largely unknown. Here, we employ single-molecule magnetic tweezers to measure, in real-time, the compaction of individual DNA molecules by the budding yeast condensin complex. We show that compaction proceeds in large (~200nm) steps, driving DNA molecules into a fully condensed state against forces of up to 2pN. Compaction can be reversed by applying high forces or adding buffer of high ionic strength. While condensin can stably bind DNA in the absence of ATP, ATP hydrolysis by the SMC subunits is required for rendering the association salt-insensitive and for subsequent compaction. Our results indicate that the condensin reaction cycle involves two distinct steps, where condensin first binds DNA through electrostatic interactions before using ATP hydrolysis to encircle the DNA topologically within its ring structure, which initiates DNA compaction. The finding that both binding modes are essential for its DNA compaction activity has important implications for understanding the mechanism of chromosome compaction.

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Molecular microscopy of DNA for genome analysis: optical mapping of restriction digests

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100136623 ◽

1995 ◽

Vol 53 ◽

pp. 50-51

Author(s):

Edward J. Huff ◽

Weiwen Cai ◽

Xinghua Hu ◽

John Huang ◽

Junping Jing ◽

...

Keyword(s):

Single Molecule ◽

Genome Analysis ◽

Optical Mapping ◽

Ccd Camera ◽

Dna Molecules ◽

Specific Sequence ◽

Single Dna Molecules ◽

Single Clone ◽

Rapid Generation ◽

Glass Surfaces

Optical microscopy of individual DNA molecules has been an interesting technique for the past 15 years, but until recently has not been useful for genome analysis. We have developed Optical Mapping an emerging single molecule approach for the rapid generation of ordered restriction maps. Many identical individual DNA molecules from a single clone are elongated and fixed onto derivatized glass surfaces, digested with a restriction enzyme which cuts the DNA wherever a specific sequence pattern is found, stained with YOYO, and imaged with a cooled CCD camera attached to an automated epi-fluorescence microscope. Images are automatically processed to correct for non-uniform illumination, remove background, locate the DNA fragments, reject objects which do not look like single DNA molecules, recognize which fragments originate from an original uncut molecule, and calculate the relative sizes of the fragments by apparent length and fluorescence intensity. Results from many molecules are combined by clustering to recognize a consistent cutting pattern. Molecules which match the pattern are averaged to improve the sizing accuracy.

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