Simultaneous Single-Molecule Force and Fluorescence Sampling of DNA Nanostructure Conformations Using Magnetic Tweezers

Kemmerich, Felix E.; Swoboda, Marko; Kauert, Dominik J.; Grieb, Maj Svea; Hahn, Steffen; Schwarz, Friedrich W.; Seidel, Ralf; Schlierf, Michael

doi:10.1021/acs.nanolett.5b03956

Cited by 62 publications

(48 citation statements)

References 38 publications

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“…Finally, the labeled ssRNA strands are annealed together in order to create dsRNA substrate. (D) A hairpin with a FRET pair [102]. Two oligonucleotides with biotin, Cy3 and Cy5 incorporated during their synthesis were annealed and then ligated together to produce a looped arm (I).…”

Section: Custom Dna Substratesmentioning

confidence: 99%

“…Complementary sequences are shown in the same color. (E) A Holliday junction is prepared by annealing four oligonucleotide strands labeled with Cy3, Cy5 and biotin [102]. A 12 nt overhang on one of the arms is used to attach the structure to a digoxigenin labeled DNA tether.…”

Section: Custom Dna Substratesmentioning

confidence: 99%

“…Another strategy for the preparation of a DNA hairpin is depicted in Figure 9D where biotin and digoxigenin anchoring ends and a pair of FRET (Főrster resonance energy transfer) dyes have been combined into one molecule [102]. This kind of substrate is particularly useful in cases where both force measurement and fluorescent tracking are desirable, such as tracking the movement of DNA motor proteins under force.…”

Section: Custom Dna Substratesmentioning

confidence: 99%

“…Another special type of substrate used in magnetic tweezers experiment is the Holliday junction [102]. This structure was created by annealing four oligonucleotides which contain four mutually complementary single stranded regions with biotin and the FRET pair located at the appropriate arms.…”

Section: Custom Dna Substratesmentioning

confidence: 99%

“…In this respect, simultaneous detection of single molecule fluorescence and deformation offers new possibilities to mechanistic studies. An example of this has been recently reported using DNA hairpin formation for the proof of concept [102].…”

Section: Integration Of Magnetic Tweezers With Fluorescence Microscopymentioning

confidence: 99%

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A Guide to Magnetic Tweezers and Their Applications

Sarkar

Rybenkov

2016

Front. Phys.

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Magnetic force spectroscopy is a rapidly developing single molecule technique that has found numerous applications at the interface of physics and biology. Since the invention of the first magnetic tweezers, a number of modifications to the approach have helped to relieve the limitations of the original design while amplifying its strengths. Inventive molecular biology solutions further advanced the technique by expanding its possible applications. In its present form, the method can be applied to both single molecules and live cells without resorting to intense irradiation, can be easily multiplexed, accommodates multiple DNAs, displays impressive resolution, and allows a remarkable ease in the stretching and twisting of macromolecules. In this review, we describe the architecture of magnetic tweezers, key requirements for experimental design and analysis of data, and outline several applications of the method that illustrate its versatility.

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Section: Custom Dna Substratesmentioning

confidence: 99%

Section: Custom Dna Substratesmentioning

confidence: 99%

Section: Custom Dna Substratesmentioning

confidence: 99%

Section: Custom Dna Substratesmentioning

confidence: 99%

Section: Integration Of Magnetic Tweezers With Fluorescence Microscopymentioning

confidence: 99%

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A Guide to Magnetic Tweezers and Their Applications

Sarkar

Rybenkov

2016

Front. Phys.

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An Introduction to Magnetic Tweezers

Dulin

2023

Methods in Molecular Biology

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Magnetic tweezers are a single-molecule force and torque spectroscopy technique that enable the mechanical interrogation in vitro of biomolecules, such as nucleic acids and proteins. They use a magnetic field originating from either permanent magnets or electromagnets to attract a magnetic particle, thus stretching the tethering biomolecule. They nicely complement other force spectroscopy techniques such as optical tweezers and atomic force microscopy (AFM) as they operate as a very stable force clamp, enabling long-duration experiments over a very broad range of forces spanning from 10 fN to 1 nN, with 1–10 milliseconds time and sub-nanometer spatial resolution. Their simplicity, robustness, and versatility have made magnetic tweezers a key technique within the field of single-molecule biophysics, being broadly applied to study the mechanical properties of, e.g., nucleic acids, genome processing molecular motors, protein folding, and nucleoprotein filaments. Furthermore, magnetic tweezers allow for high-throughput single-molecule measurements by tracking hundreds of biomolecules simultaneously both in real-time and at high spatiotemporal resolution. Magnetic tweezers naturally combine with surface-based fluorescence spectroscopy techniques, such as total internal reflection fluorescence microscopy, enabling correlative fluorescence and force/torque spectroscopy on biomolecules. This chapter presents an introduction to magnetic tweezers including a description of the hardware, the theory behind force calibration, its spatiotemporal resolution, combining it with other techniques, and a (non-exhaustive) overview of biological applications.

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