Chapter title |
DNA Origami-Based Single-Molecule Force Spectroscopy and Applications
|
---|---|
Chapter number | 23 |
Book title |
Single Molecule Analysis
|
Published by |
Humana, New York, NY, October 2023
|
DOI | 10.1007/978-1-0716-3377-9_23 |
Pubmed ID | |
Book ISBNs |
978-1-07-163376-2, 978-1-07-163377-9
|
Authors |
Kevin Kramm, Tim Schröder, Andrés Manuel Vera, Lennart Grabenhorst, Philip Tinnefeld, Dina Grohmann |
Abstract |
Over the last years, single-molecule force spectroscopy provided insights into the intricate connection between mechanical stimuli and biochemical signaling. The underlying molecular mechanisms were uncovered and explored using techniques such as atomic force microscopy and force spectroscopy using optical or magnetic tweezers. These experimental approaches are limited by thermal noise resulting from a physical connection of the studied biological system to the macroscopic world. To overcome this limitation, we recently introduced the DNA origami force clamp (FC) which is a freely diffusing nanodevice that generates piconewton forces on a DNA sequence of interest. Binding of a protein to the DNA under tension can be detected employing fluorescence resonance energy transfer (FRET) as a sensitive readout.This protocol introduces the reader to the working principles of the FC and provides instructions to design and generate a DNA origami FC customized for a protein of interest. Molecular cloning techniques are employed to modify, produce, and purify a custom DNA scaffold. A fluorescently labeled DNA suitable to detect protein binding via FRET is generated via enzymatic ligation of commercial DNA oligonucleotides. After thermal annealing of all components, the DNA origami FC is purified using agarose gel electrophoresis. The final section covers the interrogation of the FC using confocal single-molecule FRET measurements and subsequent data analysis to quantify the binding of a DNA-binding protein to its cognate recognition site under a range of forces. Using this approach, force-dependent DNA-protein interactions can be studied on the single-molecule level on thousands of molecules in a parallelized fashion. |
X Demographics
As of 1 July 2024, you may notice a temporary increase in the numbers of X profiles with Unknown location. Click here to learn more.
Geographical breakdown
Country | Count | As % |
---|---|---|
Unknown | 1 | 100% |
Demographic breakdown
Type | Count | As % |
---|---|---|
Members of the public | 1 | 100% |
Mendeley readers
Geographical breakdown
Country | Count | As % |
---|---|---|
Unknown | 1 | 100% |
Demographic breakdown
Readers by professional status | Count | As % |
---|---|---|
Student > Ph. D. Student | 1 | 100% |
Student > Bachelor | 1 | 100% |
Readers by discipline | Count | As % |
---|---|---|
Agricultural and Biological Sciences | 1 | 100% |
Chemistry | 1 | 100% |