Single-Molecule Studies of DNA, Visualization and Manipulation of Individual DNA Molecules with Fluorescence Microscopy and Optical Tweezers

2012 ◽  
Keyword(s):  
Fluorescence Microscopy ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Dna Molecules ◽  
Single Molecule Studies

scholarly journals Constructing arrays of nucleosome positioning sequences using Gibson Assembly for single-molecule studies

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Dian Spakman ◽  
Graeme A. King ◽  
Erwin J. G. Peterman ◽  
Gijs J. L. Wuite
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Building Blocks ◽  
Nucleosome Positioning ◽  
Dna Molecules ◽  
Gibson Assembly ◽  
Single Molecule Studies ◽  
Novel Method ◽  
High Degree

Abstract As the basic building blocks of chromatin, nucleosomes play a key role in dictating the accessibility of the eukaryotic genome. Consequently, nucleosomes are involved in essential genomic transactions such as DNA transcription, replication and repair. In order to unravel the mechanisms by which nucleosomes can influence, or be altered by, DNA-binding proteins, single-molecule techniques are increasingly employed. To this end, DNA molecules containing a defined series of nucleosome positioning sequences are often used to reconstitute arrays of nucleosomes in vitro. Here, we describe a novel method to prepare DNA molecules containing defined arrays of the ‘601’ nucleosome positioning sequence by exploiting Gibson Assembly cloning. The approaches presented here provide a more accessible and efficient means to generate arrays of nucleosome positioning motifs, and facilitate a high degree of control over the linker sequences between these motifs. Nucleosomes reconstituted on such arrays are ideal for interrogation with single-molecule techniques. To demonstrate this, we use dual-trap optical tweezers, in combination with fluorescence microscopy, to monitor nucleosome unwrapping and histone localisation as a function of tension. We reveal that, although nucleosomes unwrap at ~20 pN, histones (at least histone H3) remain bound to the DNA, even at tensions beyond 60 pN.

DNA visualization in single molecule studies carried out with optical tweezers: Covalent versus non-covalent attachment of fluorophores

2015 ◽  
Vol 466 (2) ◽  
pp. 226-231 ◽  
Author(s):  
Sandy Suei ◽  
Allan Raudsepp ◽  
Lisa M. Kent ◽  
Stephen A.J. Keen ◽  
Vyacheslav V. Filichev ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Covalent Attachment ◽  
Single Molecule Studies

scholarly journals Profound Nanoscale Structural and Biomechanical Changes in DNA Helix upon Treatment with Anthracycline Drugs

2020 ◽  
Vol 21 (11) ◽  
pp. 4142
Author(s):  
Aleksandra Kaczorowska ◽  
Weronika Lamperska ◽  
Kaja Frączkowska ◽  
Jan Masajada ◽  
Sławomir Drobczyński ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Regulatory Elements ◽  
Dna Molecules ◽  
Anthracycline Antibiotics ◽  
Single Molecule Level ◽  
Force Microscopy ◽  
Atomic Force ◽  
Microscopy Analysis ◽  
Dna Regulatory Elements

In our study, we describe the outcomes of the intercalation of different anthracycline antibiotics in double-stranded DNA at the nanoscale and single molecule level. Atomic force microscopy analysis revealed that intercalation results in significant elongation and thinning of dsDNA molecules. Additionally, using optical tweezers, we have shown that intercalation decreases the stiffness of DNA molecules, that results in greater susceptibility of dsDNA to break. Using DNA molecules with different GC/AT ratios, we checked whether anthracycline antibiotics show preference for GC-rich or AT-rich DNA fragments. We found that elongation, decrease in height and decrease in stiffness of dsDNA molecules was highest in GC-rich dsDNA, suggesting the preference of anthracycline antibiotics for GC pairs and GC-rich regions of DNA. This is important because such regions of genomes are enriched in DNA regulatory elements. By using three different anthracycline antibiotics, namely doxorubicin (DOX), epirubicin (EPI) and daunorubicin (DAU), we could compare their detrimental effects on DNA. Despite their analogical structure, anthracyclines differ in their effects on DNA molecules and GC-rich region preference. DOX had the strongest overall effect on the DNA topology, causing the largest elongation and decrease in height. On the other hand, EPI has the lowest preference for GC-rich dsDNA. Moreover, we demonstrated that the nanoscale perturbations in dsDNA topology are reflected by changes in the microscale properties of the cell, as even short exposition to doxorubicin resulted in an increase in nuclei stiffness, which can be due to aberration of the chromatin organization, upon intercalation of doxorubicin molecules.

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

2004 ◽  
Vol 18 (2) ◽  
pp. 203-211 ◽  
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.

scholarly journals Dissecting Elastic Heterogeneity along DNA Molecules Coated Partly with Rad51 Using Concurrent Fluorescence Microscopy and Optical Tweezers

2006 ◽  
Vol 91 (8) ◽  
pp. L78-L80 ◽  
Author(s):  
Joost van Mameren ◽  
Mauro Modesti ◽  
Roland Kanaar ◽  
Claire Wyman ◽  
Gijs J.L. Wuite ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Optical Tweezers ◽  
Dna Molecules
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