DNA-protein interactions are fascinating. It’s difficult to do, but understanding these interactions opens the door to a lot of medical innovation. U.S. Department of Energy’s Lawrence Berkeley National Laboratory is taking a new approach in this field by using plasmon resonance to measure the exact point in nanometers between proteins and DNA. Here’s how the “ruler” works:
The molecular ruler was developed by a team of scientists that includes UC Berkeley Bioengineering Professor Luke Lee, UC Berkeley Ph.D. student Gang Liu, and Paul Alivisatos, a Berkeley Lab chemist in the Materials Sciences Division and an Associate Laboratory Director. It’s composed of gold nanoparticles that are coated with a substance that makes the nanoparticles soluble. Next, about 100 double-stranded DNA segments are tethered to the gold nanoparticle in a configuration that resembles a many-legged spider.
The ruler works because of plasmon resonance, which is the collection of electrons that resonate in a metallic particle, in this case the gold-DNA conjugate. Plasmon resonance changes as a particle changes, leading to differences in scattering wavelength. For example, if the gold particle’s spidery DNA strands, which are 54 base pairs long, are shortened for whatever reason, then the gold-DNA particle’s scattering wavelengths also shift – and this shift can be easily detected using spectroscopy. This method is so sensitive that scientists can use it to detect whether a DNA strand has been shortened by as little as one base pair in length, which opens the door for mapping the exact location of protein-DNA interactions.
Chen and colleagues put the ruler to the test by using it to conduct DNA footprinting, a process in which scientists identify where on a DNA strand a particular protein attaches itself. DNA footprinting is most commonly performed on proteins that are thought to play a significant functional role, such as in regulating gene expression.
To conduct this genetic sleuthing, they developed a customized gold-DNA conjugate. As usual, they attached to each gold nanoparticle roughly 100 DNA strands that are 54 base pairs long. But among these base pairs they inserted a sequence of six base pairs that are specially tailored to bind to a model protein, in this case EcoRI(Q111). In other words, at the same location on each strand, they encoded the perfect home for an EcoRI(Q111) protein. They introduced this protein to the specially prepared gold-DNA conjugates, and allowed the protein to bind to the DNA strands.
Next, to map exactly where the protein attaches to the DNA, they introduced an enzyme called an exonuclease. This enzyme clamps onto the free end of the DNA strands, and chomps down each strand, removing base pair after base pair, until it’s blocked by the recently attached EcoRI(Q111) protein. It’s like someone slurping down a spaghetti noodle, only to be stopped cold by a fly sitting on the noodle.
In this way, the gold particles’s DNA strands are shortened, with their newly sheared free ends marking the location of the protein. And this, in turn, allows the research team to zero in on the DNA’s protein binding site. They already know the plasmonic scattering signature of the gold-DNA particle with all of its 54 base pairs. Now, they can then measure the plasmonic signature of the gold-DNA particle after its DNA has been trimmed. The difference between the two spectra correlates to the number of base pairs eliminated by the exonuclease.
Check out the press release from Lawrence Berkeley National Laboratory here…