We describe an approach to detect the frequency of interaction between any two genomic loci. Generation of a matrix of interaction frequencies between sites on the same or different chromosomes reveals their relative spatial disposition and provides information about the physical properties of the chromatin fiber. This methodology can be applied to the spatial organization of entire genomes in organisms from bacteria to human. Using the yeast Saccharomyces cerevisiae, we could confirm known qualitative features of chromosome organization within the nucleus and dynamic changes in that organization during meiosis. We also analyzed yeast chromosome III at the G1 stage of the cell cycle. We found that chromatin is highly flexible throughout. Furthermore, functionally distinct AT- and GC-rich domains were found to exhibit different conformations, and a population-average 3D model of chromosome III could be determined. Chromosome III emerges as a contorted ring.

/pdf/capturing-chromosome-conformation-prwxnw8moe.pdf

Capturing Chromosome Conformation

http://experimentationlab.berkeley.edu/sites/default/files/AFMImages/butt_AFM.pdf

Force measurements with the atomic force microscope: Technique, interpretation and applications

The specific bonding of DNA base pairs provides the chemical foundation for genetics. This powerful molecular recognition system can be used in nanotechnology to direct the assembly of highly structured materials with specific nanoscale features, as well as in DNA computation to process complex information. The exploitation of DNA for material purposes presents a new chapter in the history of the molecule.

/pdf/dna-in-a-material-world-28gymklany.pdf

DNA in a material world

Single-molecule force spectroscopy has emerged as a powerful tool to investigate the forces and motions associated with biological molecules and enzymatic activity. The most common force spectroscopy techniques are optical tweezers, magnetic tweezers and atomic force microscopy. Here we describe these techniques and illustrate them with examples highlighting current capabilities and limitations.

Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy

Molecular recognition between complementary strands of DNA allows construction on a nanometre length scale. For example, DNA tags may be used to organize the assembly of colloidal particles, and DNA templates can direct the growth of semiconductor nanocrystals and metal wires. As a structural material in its own right, DNA can be used to make ordered static arrays of tiles, linked rings and polyhedra. The construction of active devices is also possible--for example, a nanomechanical switch, whose conformation is changed by inducing a transition in the chirality of the DNA double helix. Melting of chemically modified DNA has been induced by optical absorption, and conformational changes caused by the binding of oligonucleotides or other small groups have been shown to change the enzymatic activity of ribozymes. Here we report the construction of a DNA machine in which the DNA is used not only as a structural material, but also as 'fuel'. The machine, made from three strands of DNA, has the form of a pair of tweezers. It may be closed and opened by addition of auxiliary strands of 'fuel' DNA; each cycle produces a duplex DNA waste product.

A DNA-fuelled molecular machine made of DNA

Single molecules of double-stranded DNA (dsDNA) were stretched with force-measuring laser tweezers. Under a longitudinal stress of approximately 65 piconewtons (pN), dsDNA molecules in aqueous buffer undergo a highly cooperative transition into a stable form with 5.8 angstroms rise per base pair, that is, 70% longer than B form dsDNA. When the stress was relaxed below 65 pN, the molecules rapidly and reversibly contracted to their normal contour lengths. This transition was affected by changes in the ionic strength of the medium and the water activity or by cross-linking of the two strands of dsDNA. Individual molecules of single-stranded DNA were also stretched giving a persistence length of 7.5 angstroms and a stretch modulus of 800 pN. The overstretched form may play a significant role in the energetics of DNA recombination.

/pdf/overstretching-b-dna-the-elastic-response-of-individual-16c6cujz0e.pdf

Overstretching B-DNA: The Elastic Response of Individual Double-Stranded and Single-Stranded DNA Molecules

Single chicken erythrocyte chromatin fibers were stretched and released at room temperature with force-measuring laser tweezers. In low ionic strength, the stretch-release curves reveal a process of continuous deformation with little or no internucleosomal attraction. A persistence length of 30 nm and a stretch modulus of ≈5 pN is determined for the fibers. At forces of 20 pN and higher, the fibers are modified irreversibly, probably through the mechanical removal of the histone cores from native chromatin. In 40–150 mM NaCl, a distinctive condensation-decondensation transition appears between 5 and 6 pN, corresponding to an internucleosomal attraction energy of ≈2.0 kcal/mol per nucleosome. Thus, in physiological ionic strength the fibers possess a dynamic structure in which the fiber locally interconverting between “open” and “closed” states because of thermal fluctuations.

https://www.pnas.org/content/pnas/97/1/127.full.pdf

Yujia Cui

Papers

Overstretching B-DNA: The Elastic Response of Individual Double-Stranded and Single-Stranded DNA Molecules

Pulling a single chromatin fiber reveals the forces that maintain its higher-order structure