Sample preparation method to improve the efficiency of high-throughput single-molecule force spectroscopy

Jin, Lei; Li, Kou; Zeng, Yanan; Hu, Chunguang; Hu, Xiaodong

doi:10.1007/s41048-019-00097-4

Cited by 2 publications

(3 citation statements)

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“…In order to lift this uncertainty, one may eventually have recourse to more efficient sample preparation techniques, which do not involve hydrophilic patches to which streptavidincoated NPs may adhere. 58 Range of DNA Concentrations Available with the TPM Setup. In connection with the previous point, BD simulations suggest that the sole addition of globular crowders in a standard TPM setup will probably not be sufficient to investigate the range of DNA concentration that prevails in prokaryotic cells.…”

Section: ■ Discussionmentioning

confidence: 99%

“…The problem is that it is actually difficult to discriminate experimentally between NPs which are immobilized because they adhere to the coating and NPs which are maintained at a short distance of the bottom surface by collapsed DNA molecules. In order to lift this uncertainty, one may eventually have recourse to more efficient sample preparation techniques, which do not involve hydrophilic patches to which streptavidin-coated NPs may adhere …”

Section: Discussionmentioning

confidence: 99%

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Tethered Particle Motion Technique in Crowded Media: Compaction of DNA by Globular Macromolecules

Joyeux

2024

J. Phys. Chem. B

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Tethered Particle Motion (TPM) is a single molecule technique, which consists in tracking the motion of a nanoparticle (NP) immersed in a fluid and tethered to a glass surface by a DNA molecule. The present work addresses the question of the applicability of TPM to fluids which contain crowders at volume fractions ranging from that of the nucleoid of living bacteria (around 30%) up to the jamming threshold (around 66%). In particular, we were interested in determining whether TPM can be used to characterize the compaction of DNA by globular crowders. To this end, extensive Brownian Dynamics simulations were performed with a specifically built coarse-grained model. Analysis of the simulations reveals several effects not observed in dilute media, which impose constraints on the TPM setup. In particular, the Tethered Fluorophore Motion (TFM) technique, which consists in replacing the NP by a much smaller fluorophore, is probably better suited than standard TPM. Moreover, a sample preparation technique which does not involve hydrophilic patches may be required. Finally, the use of a DNA brush may be needed to achieve DNA concentrations close to in vivo ones.

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Section: ■ Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Tethered Particle Motion Technique in Crowded Media: Compaction of DNA by Globular Macromolecules

Joyeux

2024

J. Phys. Chem. B

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“…The Forde lab developed a miniradio CFM that used wireless radio frequency transmission for a plug-and-play CFM module (9). The Hu lab developed a CFM that incorporated digital holography for 3D particle tracking (10).…”

Section: Introductionmentioning

confidence: 99%

A Wi-Fi live streaming Centrifuge Force Microscope for benchtop single-molecule experiments

Punnoose

Hayden

Zhou

et al. 2020

Preprint

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AbstractThe ability to apply controlled forces to individual molecules has been revolutionary in shaping our understanding of biophysics in areas as diverse as dynamic bond strength, biological motor operation, and DNA replication. However, the methodology to perform single-molecule experiments has been and remains relatively inaccessible due to cost and complexity. In 2010, we introduced the Centrifuge Force Microscope (CFM) as a new platform for accessible and high-throughput single-molecule experimentation. The CFM consists of a rotating microscope where prescribed centrifugal forces can be applied to microsphere-tethered biomolecules. In this work, we develop and demonstrate a next-generation Wi-Fi CFM that offers unprecedented ease of use and flexibility in design. The modular CFM unit fits within a standard benchtop centrifuge and connects by Wi-Fi to a external computer for live control and streaming at near gigabit speeds. The use of commercial wireless hardware allows for flexibility in programming and provides a streamlined upgrade path as Wi-Fi technology improves. To facilitate ease of use, detailed build and setup instructions are provided, as well as LabVIEW™ based control software and MATLAB® based analysis software. We demonstrate the analysis of force-dependent dissociation of short DNA duplexes of 7, 8, and 9 bp using the instrument. We showcase the sensitivity of the approach by resolving distinct dissociation kinetic rates for a 7 bp duplex where one G-C base pair is mutated to an A-T base pair.SignificanceThe ability to apply mechanical forces to individual molecules has provided unprecedented insight into many areas of biology. Centrifugal force provides a way to increase the throughput and to decrease the cost and complexity of single-molecule experiments compared to other approaches. In this work, we develop and demonstrate a new user-friendly Centrifuge Force Microscope (CFM) that enables live-streaming of high-throughput single-molecule experiments in a benchtop centrifuge. We achieved near gigabit bandwidth with standard Wi-Fi components, and we provide detailed design instructions and software to facilitate use by other labs. We demonstrate the instrument for sensitive kinetic measurements that are capable of resolving the difference between two DNA duplexes that differ by a single G-C to A-T substitution.

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Sample preparation method to improve the efficiency of high-throughput single-molecule force spectroscopy

Cited by 2 publications

References 30 publications

Tethered Particle Motion Technique in Crowded Media: Compaction of DNA by Globular Macromolecules

Tethered Particle Motion Technique in Crowded Media: Compaction of DNA by Globular Macromolecules

A Wi-Fi live streaming Centrifuge Force Microscope for benchtop single-molecule experiments

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