Quadruple optical trap unravels bacterial DNA
Research reveals information about DNA compaction
Bacteria have no nucleus, and these “simpler” organisms have been a challenge for researchers looking to better understand how bacterial DNA is compacted and organized in the cell. Now, thanks to a specially designed optical trap, researchers at Vrije University in Amsterdam, the Netherlands, understand more about how a linking protein, histonelike nucleoid structuring protein (H-NS), bridges two strands of DNA. Long-term, the research may lead to new drugs for fighting bacterial infection.
Uncompacted DNA is very long. So, to squeeze it into a cell, various proteins -- such as H-NS, leucine-responsive regulatory protein or structure maintenance of chromosome protein -- catch hold of it and fold it into hundreds of loops. Scientists believe that by bridging DNA duplexes, H-NS is important for organizing and compacting bacterial chromatin. They also believe that the protein regulates gene expression and may play a role in the bacteria’s ability to adapt to their environment and their virulence.
Remus Dame of the university’s physics department said that little research has been done to explore how H-NS functions. He explained that biochemical work has shown that some linking proteins cause DNA molecules in solution to aggregate. In addition, electron microscopy and scanning force microscopy have made detailed images of the complexes formed by DNA and linking proteins. “In the case of H-NS, we demonstrated by scanning force microscopy that DNA loops back onto itself or bridges multiple molecules,” he added. “None of these methods gives really detailed insight into the nature of the interaction and its dynamics.”
Dame said the group studied the H-NS bond because it is essential to DNA organization and compaction and because only static structural data was available for it. To really understand the nature of the forces that the protein exerts to hold DNA strands together or to fold DNA upon itself, he and his colleagues, Maarten Noom and Gijs Wuite, wanted to determine the force necessary to break the bonds. To do this, they developed a special optical trapping setup that allows them to trap and manipulate four polystyrene beads.
To conduct the experiment, the researchers attached the beads to the ends of two DNA molecules. Then they captured two DNA molecules and introduced them to an H-NS solution, which quickly linked the two molecules together. Leaving one end of the joined DNA molecules stationary, they moved the remaining two optical traps apart, breaking the bonds successively like unzipping a zipper.
Two strands bound
Dame said that they chose to bind two DNA strands together to provide clearer information on binding. “One can, in principle, think of doing a similar experiment with only one DNA molecule -- as in traditional single-molecule stretching experiments with optical or magnetic tweezers,” he said. Using the H-NS to fold only one strand of DNA would create multiple loops that also probably would interact with themselves. “If one disrupts these loops by pulling, it will not be clear what type of complex is actually disrupted and the ‘type of force’ exerted is also unclear,” he added.
Using a custom-built optical trap with four separate traps, researchers have gained a better understanding of a key protein that folds bacterial DNA into a package small enough to be contained in the cell wall. Reprinted with permission of Nature.
As reported in the Nov. 16 issue of Nature, the researchers created the four optical traps, which they call the Q-trap, with an Nd:YVO4 laser from Spectra-Physics. The CW laser produced 10 W at 1064 nm. A Faraday isolator from Optics for Research protected the beam against backreflections. They also used a beam expander from Linos Photonics GmbH. To create the four traps, the beam was split, with one beam path going through an acousto-optic deflector from IntraAction Corp., which allowed the same beam to be time-shared over three locations to create three optical traps. Two orthogonally placed acousto-optic deflectors allowed steering of the traps in both directions in the sample plane. The researchers coupled the first-order deflected beam along both axes into a 60× water-immersion objective from Nikon in a custom-built microscope.
The other beam path entered a 1:1 telescope system that allowed the group to steer it in the sample and to generate a continuous optical trap. LabView software enabled location control.
To detect the displacement of the continuous trap, the researchers imaged the intensity profile in the back focal plane of the condenser onto a quadrant photodiode from UDT Sensors. Measuring the end-to-end distance required determining the distance between the stationary and the moving bead from video microscopy and analyzing it with LabView.
The force required to break the bonds varied with the speed of unzipping, with slower speeds producing lower breaking forces. For example, when the beads were moved apart at 6.5 nm/s, the breaking force was about 1 pN. However, increasing the speed to 88 nm/s brought the force required to break the bond to about 25 pN. The force needed to break a bond, however, is not enough to impede transcription. Studies have shown that RNA polymerase can generate forces as high as 25 pN.
In addition, the researchers discovered that, as the duplex DNA was pulled apart, the force displayed steps that likely corresponded to the breaking of individual H-NS bonds. By fitting the force data to a histogram, the group determined that the steps fit well to multiple Gaussians with identical spacing of 3.6 ±0.4 nm, which is the same spacing as the helical repeat for B-DNA.
“The Q-trap has opened up the possibility of new types of experiments,” Dame said. “Currently, there are no other methods for getting similar information or having this degree of control in analyzing the protein-DNA complex.”
He said the group plans to conduct similar experiments with the large number of biologically relevant proteins that can link DNA molecules. “Besides determining the basic properties of these proteins, we intend to include motor proteins, such as RNA polymerase, in our dual DNA experiments to determine the effects of roadblocks [such as linked DNA] on motor protein progression.”
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