If I told you that my summer work involves game controllers, lasers, and avoiding obstacles, you might not believe that I am working in a biophysics research lab. But it turns out that, in Ariel Kaplan’s laboratory at the Israel Institute of Technology (Technion), such video-game-like setups are used to study DNA. This Haifa-based lab uses X-box controllers to manipulate a laser-based technology called optical tweezers to trap DNA using laser beams (while avoiding air bubbles, which threaten to displace the DNA that is trapped in the beams). This action-packed technology is useful because it lets us measure the forces that bind DNA to proteins. This data can enhance our physical understanding of protein-DNA complexes, many of which carry crucial structural and functional implications.
DNA faces a unique challenge. On one hand, this hereditary molecule must be compacted enough so that it can fit into the nucleus of a cell. The DNA in a single cell, if fully unwound, would span 3 meters (Chen), yet it must be packaged into a nucleus with a 6 micrometer radius (6 x 10-6 meters) (“Diameter of a Spermocyte Nucleus”).This is equivalent to taking a thin string that stretches from the ground to the top of the empire state building, and coiling it up so that it fits inside a sesame seed! At the same time, the genetic information encoded in the DNA must be accessible to transcription factors (TFs), proteins that initiate the transcription of DNA into RNA (which in turn directly codes for proteins). There is a trade-off between these two requirements: the more a sequence of DNA is packaged, the harder for TFs to bind to the sequence (Goodsell). The Kaplan lab aims to gain a more complete picture of how DNA can simultaneously achieve sufficient compaction and proper transcription.
To investigate this question, the Kaplan lab uses optical tweezers to study the most basic DNA packaging unit, called a nucleosome. A nucleosome is a bead-like complex consisting of DNA wrapped around a group of 8 proteins known as histones (Goodsell). The Kaplan lab isolates fragments of DNA believed to contain nucleosomes, and traps them using optical tweezers. The tweezers then exert opposing forces on each of DNA’s two strands, causing the strands to separate. If the DNA is part of a nucleosome, then it takes significantly more force to separate the strands than if the DNA is not part of such a complex. This is because nucleosomes form stabilizing interactions with double-stranded DNA, making it harder to break. Thus, by measuring the force required to unravel a DNA fragment, we can infer whether or not that fragment interacts with a histone. We can also gauge how strong the interaction is, as a tighter DNA-histone association requires more force to break than a weaker one (Ming and Wang).
This is equivalent to taking a thin string that stretches from the ground to the top of the empire state building, and coiling it up so that it fits inside a sesame seed!”
Given that association with histones comes at the expense of association with transcription factors, it seems reasonable to hypothesize that DNA-histone interactions decrease in strength when there is a need for DNA transcription. Our next goal in the investigation is to test this hypothesis. We can set up two experimental conditions, one consisting of DNA samples with nucleosomes and TFs, and the other consisting of DNA with only nucleosomes. Using optical tweezers, we can determine the force needed to unwind sequences from each of these conditions, and compare the force “profiles”. We are especially interested in sequence regions called promoters, which are known to bind TFs (in addition to histones). If our hypothesis is correct, then we might find that promoter sequences require less force to unravel in the presence of TFs than they do in absence of TFs. This could suggest that, when TFs are present, DNA-histone interactions weaken, ensuring that the promoter can bind the TFs and lead to proper transcription.
How exactly do lasers come into play? After isolating a DNA sequence of interest, we chemically attach a microscopic bead to each of its two ends. We then place each bead in a laser beam. It turns out that the electric field in the laser exerts a force on the bead such that it is trapped at the center of the beam. Just as stretching or compressing a spring requires force, so does moving the bead away from its equilibrium position (this analogy is more meaningful than you may think—mathematically speaking, this property of springs and optical traps is described using the same equation in both cases) (Bakken and Koerner). When we move the laser beams apart from each other, the bead-linked DNA’s trapped ends also move apart due to the force exerted by the laser’s electric field, and so the DNA strands separate. This technique lets us quantify the force that is required to separate the strands (thanks to the spring-like relation between bead displacement and force). Such measurements may provide us with a clearer picture of the complex interactions between DNA and proteins.
- Bakken, Trygve, and Koerner, Adam. “Optical Tweezers.” UC San Diego Department of Physics. N.p., June 2009. Web. 9 July 2014. <https://physics.ucsd.edu/neurophysics/courses/physics_173_273/Optical_Tweezers_June09_Bakken_Koerner.pdf>.
- Chen, Steven. “Length of a Human DNA Molecule.” The Physics Factbook. N.p., n.d. Web. 09 July 2014. <http://hypertextbook.com/facts/1998/StevenChen.shtml>.
- “Diameter of Spermatocyte Nucleus.” Bionumbers. Harvard Medical School, 8 June 2009. Web. 9 July 2014.
- Goodsell, David. “Nucleosome.” RCSB Protein Data Bank. N.p., July 2000. Web. 09 July 2014.
- Li, Ming, and Michelle D. Wang. “Methods in Enzymology.” Unzipping Single DNA Molecules to Study Nucleosome Structure and Dynamics513 (2012): 29-58. Print.
Amir Bitran is a Brevia editor. He can be reached at email@example.com.