Resonator nanophotonic standing-wave array trap for single-molecule manipulation and measurement

AbstractNanophotonic tweezers represent emerging platforms with significant potential for parallel manipulation and measurements of single biological molecules on-chip. However, trapping force generation represents a substantial obstacle for their broader utility. Here, we present a resonator nanophotonic standing-wave array trap (resonator-nSWAT) that demonstrates significant force enhancement. This platform integrates a critically-coupled resonator design to the nSWAT and incorporates a novel trap reset scheme. The nSWAT can now perform standard single-molecule experiments, including stretching DNA molecules to measure their force-extension relations, unzipping DNA molecules, and disrupting and mapping protein-DNA interactions. These experiments have realized trapping forces on the order of 20 pN while demonstrating base-pair resolution with measurements performed on multiple molecules in parallel. Thus, the resonator-nSWAT platform now meets the benchmarks of a table-top precision optical trapping instrument in terms of force generation and resolution. This represents the first demonstration of a nanophotonic platform for such single-molecule experiments.

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Single molecule force spectroscopy studies of DNA denaturation by T4 gene 32 protein

Spectroscopy An International Journal ◽

10.1155/2004/403203 ◽

2004 ◽

Vol 18 (2) ◽

pp. 203-211 ◽

Cited By ~ 16

Author(s):

Mark C. Williams ◽

Kiran Pant ◽

Ioulia Rouzina ◽

Richard L. Karpel

Keyword(s):

Dna Binding ◽

Optical Tweezers ◽

Single Molecule ◽

Protein Interactions ◽

Force Spectroscopy ◽

Melting Transition ◽

Dna Molecules ◽

Force Extension ◽

Single Molecule Force Spectroscopy ◽

Gene 32

Single molecule force spectroscopy is an emerging technique that can be used to measure the biophysical properties of single macromolecules such as nucleic acids and proteins. In particular, single DNA molecule stretching experiments are used to measure the elastic properties of these molecules and to induce structural transitions. We have demonstrated that double‒stranded DNA molecules undergo a force‒induced melting transition at high forces. Force–extension measurements of single DNA molecules using optical tweezers allow us to measure the stability of DNA under a variety of solution conditions and in the presence of DNA binding proteins. Here we review the evidence of DNA melting in these experiments and discuss the example of DNA force‒induced melting in the presence of the single‒stranded DNA binding protein T4 gene 32. We show that this force spectroscopy technique is a useful probe of DNA–protein interactions, which allows us to obtain binding rates and binding free energies for these interactions.

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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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Nanometric plasmonic optical trapping on gold nanostructures

The European Physical Journal Applied Physics ◽

10.1051/epjap/2019180347 ◽

2019 ◽

Vol 86 (3) ◽

pp. 30501

Author(s):

Domna G. Kotsifaki ◽

Mersini Makropoulou ◽

Alexander A. Searfetinides

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Optical Trapping ◽

Numerical Aperture ◽

Resonance Wavelength ◽

Objective Lens ◽

Plasmonic Nanostructures ◽

Theoretical Support ◽

Trapping Force ◽

Trapping Forces

The precise noninvasive optical manipulation of nanometer-sized particles by evanescent fields, instead of the conventional optical tweezers, has recently awaken an increasing interest, opening a way for investigating phenomena relevant to both fundamental and applied science. In this work, the optical trapping force exerted on trapped dielectric nanoparticle was theoretically investigated as a function on the trapping beam wavelength and as a function of several plasmonic nanostructures schemes based on numerical simulation. The maximum optical trapping forces are obtained at the resonance wavelength for each plasmonic nanostructure geometry. Prominent tunabilities, such as radius and separation of gold nanoparticles as well as the numerical aperture of objective lens were examined. This work will provide theoretical support for developing new types of plasmonic sensing substrates for exciting biomedical applications such as single-molecule fluorescence.

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Miniaturized Millimeter-Wave On-Chip Resonator Design in Silicon Technology

2020 IEEE International Conference on Integrated Circuits, Technologies and Applications (ICTA) ◽

10.1109/icta50426.2020.9332098 ◽

2020 ◽

Author(s):

Ling Wang ◽

Ying Jiang ◽

Zhenhai Zhang ◽

Sicheng Yu

Keyword(s):

Millimeter Wave ◽

Silicon Technology ◽

Resonator Design ◽

On Chip

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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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Ripping RNA by Force using Gaussian Network Models

10.1101/075820 ◽

2016 ◽

Author(s):

Changbong Hyeon ◽

D. Thirumalai

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Cross Correlation ◽

Network Models ◽

Force Extension ◽

Correlation Matrices ◽

Rna Molecules ◽

Allosteric Communication ◽

Ion Effects ◽

Gaussian Network

AbstractUsing force as a probe to map the folding landscapes of RNA molecules has become a reality thanks to major advances in single molecule pulling experiments. Although the unfolding pathways under tension are complicated to predict studies in the context of proteins have shown that topology plays is the major determinant of the unfolding landscapes. By building on this finding we study the responses of RNA molecules to force by adapting Gaussian network model (GNM) that represents RNAs using a bead-spring network with isotropic interactions. Cross-correlation matrices of residue fluctuations, which are analytically calculated using GNM even upon application of mechanical force, show distinct allosteric communication as RNAs rupture. The model is used to calculate the force-extension curves at full thermodynamic equilibrium, and the corresponding unfolding pathways of four RNA molecules subject to a quasi-statically increased force. Our study finds that the analysis using GNM captures qualitatively the unfolding pathway of T. ribozyme elucidated by the optical tweezers measurement. However, the simple model is not sufficient to capture subtle features, such as bifurcation in the unfolding pathways or the ion effects, in the forced-unfolding of RNAs.

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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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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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