Atomic force microscopy-based single-molecule force spectroscopy detects DNA base mismatches

Nanoscale ◽  
2019 ◽  
Vol 11 (37) ◽  
pp. 17206-17210 ◽  
Author(s):  
Wenjing Liu ◽  
Yourong Guo ◽  
Kaizhe Wang ◽  
Xingfei Zhou ◽  
Ying Wang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Force Spectroscopy ◽  
Dna Origami ◽  
Single Molecule Force Spectroscopy ◽  
Force Microscopy ◽  
Atomic Force ◽  
Base Pair Mismatch ◽  
Dna Base ◽  
Single Base Pair ◽  
Target Molecules

AFM-based single-molecule-force spectroscopy is limited by low throughput. We introduce addressable DNA origami to study multiple target molecules at once. Target DNAs differing by only a single-base pair mismatch are clearly differentiated.

Reverse Engineering of an Affinity-Switchable Molecular Interaction Characterized by Atomic Force Microscopy Single-Molecule Force Spectroscopy†

Langmuir ◽  
2008 ◽  
Vol 24 (4) ◽  
pp. 1365-1370 ◽  
Author(s):  
Dario Anselmetti ◽  
Frank Wilco Bartels ◽  
Anke Becker ◽  
Björn Decker ◽  
Rainer Eckel ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Reverse Engineering ◽  
Single Molecule ◽  
Molecular Interaction ◽  
Force Spectroscopy ◽  
Single Molecule Force Spectroscopy ◽  
Force Microscopy ◽  
Atomic Force

Going Vertical To Improve the Accuracy of Atomic Force Microscopy Based Single-Molecule Force Spectroscopy

ACS Nano ◽  
2017 ◽  
Vol 12 (1) ◽  
pp. 198-207 ◽  
Author(s):  
Robert Walder ◽  
William J. Van Patten ◽  
Ayush Adhikari ◽  
Thomas T. Perkins
Keyword(s):  
Atomic Force Microscopy ◽  
Single Molecule ◽  
Force Spectroscopy ◽  
Single Molecule Force Spectroscopy ◽  
Force Microscopy ◽  
Atomic Force

Single-Molecule Force Spectroscopy onBombyx moriSilk Fibroin by Atomic Force Microscopy

Langmuir ◽  
2000 ◽  
Vol 16 (9) ◽  
pp. 4305-4308 ◽  
Author(s):  
Wenke Zhang ◽  
Qiaobing Xu ◽  
Shan Zou ◽  
Hongbin Li ◽  
Weiqing Xu ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Single Molecule ◽  
Force Spectroscopy ◽  
Single Molecule Force Spectroscopy ◽  
Force Microscopy ◽  
Atomic Force

scholarly journals Bioconjugation Strategies for Connecting Proteins to DNA-Linkers for Single-Molecule Force-Based Experiments

Nanomaterials ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 2424
Author(s):  
Lyan M. van der Sleen ◽  
Katarzyna M. Tych
Keyword(s):  
Mechanical Properties ◽  
Atomic Force Microscopy ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Short Range ◽  
Force Spectroscopy ◽  
Magnetic Tweezers ◽  
Single Molecule Force Spectroscopy ◽  
Force Microscopy ◽  
Atomic Force

The mechanical properties of proteins can be studied with single molecule force spectroscopy (SMFS) using optical tweezers, atomic force microscopy and magnetic tweezers. It is common to utilize a flexible linker between the protein and trapped probe to exclude short-range interactions in SMFS experiments. One of the most prevalent linkers is DNA due to its well-defined properties, although attachment strategies between the DNA linker and protein or probe may vary. We will therefore provide a general overview of the currently existing non-covalent and covalent bioconjugation strategies to site-specifically conjugate DNA-linkers to the protein of interest. In the search for a standardized conjugation strategy, considerations include their mechanical properties in the context of SMFS, feasibility of site-directed labeling, labeling efficiency, and costs.

scholarly journals Monomeric streptavidin: a versatile regenerative handle for force spectroscopy

2018 ◽  
Author(s):  
Magnus S. Bauer ◽  
Lukas F. Milles ◽  
Steffen M. Sedlak ◽  
Hermann E. Gaub
Keyword(s):  
Single Molecule ◽  
Force Spectroscopy ◽  
Protein Domain ◽  
Expression And Purification ◽  
Single Molecule Force Spectroscopy ◽  
Site Specific ◽  
Force Microscopy ◽  
Loading Rates ◽  
Atomic Force ◽  
C Terminus

AbstractMost avidin-based handles in force spectroscopy are tetravalent biotin binders. Tetravalency presents two issues: multiple pulling geometries as well as multiple targets bound simultaneously. Additionally, such tetravalent handles require elaborate purification protocols in order to reassemble. A stoichiometric, monomeric variant of streptavidin (mcSA2) had been engineered previously. It is readily expressed and purified, and it binds biotin with a nanomolar KD. For atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS), we fused the monomeric streptavidin with a small protein domain as an experimental fingerprint and to improve solubility. A ybbR-tag was additionally included for covalent site-specific tethering. Rupture forces of the mcSA2:biotin complex were found to be in a comparable range above 150 pN at force loading rates of 1E4 pN/s as for previously published, tetravalent streptavidin:biotin systems. Additionally, when tethering mcSA2 from its C-terminus, rupture forces were found to be slightly higher than when tethered N-terminally. Due to its monomeric nature, mcSA2 could also be chemically denatured and subsequently refolded - and thus regenerated during an experiment, in case the handle gets misfolded or clogged. We show that mcSA2 features a straightforward expression and purification with flexible tags, high stability, regeneration possibilities and an unambiguous pulling geometry. Combined, these properties establish mcSA2 as a reliable handle for single-molecule force spectroscopy.

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