In our last Lab Report, we explored how parents pass on their genetic information, or DNA sequences, to their offspring by providing them with the proper set of chromosomes. This week, we will take a slightly more complex look at the cell by journeying into the lab of Candice Etson, Assistant Professor of Physics. Etson uses single-molecule imaging as a tool for analyzing interactions between DNA and proteins, the puppeteers that control the body as long as it’s alive.
“Inside your cells, you have this DNA, which is this blueprint for you,” Etson said. “Every cell has a copy of that book, and that book is constantly being damaged, so it constantly needs to be repaired. It also needs to be copied a lot of times. At the same time, there are proteins that are responsible for taking that message and turning it into [new] proteins.”
Etson elaborated on the importance of the interaction between DNA and protein.
“We don’t have every single gene being expressed all the time, so it’s protein–DNA interactions that determine which genes are going to be expressed at different times,” she said. “All of that is driven by protein-DNA interactions.”
Etson explained her background in single-molecule imaging.
“My specialty, in terms of the way I do single molecule imaging, is a technique called ‘Total Internal Reflection Fluorescence (TIRF) Microscopy,’” she said. “In this technique, you [image only] a really, really thin layer that’s very close to the cover slip in the reaction chamber that you’re [observing]. If you attach some parts of your experiment—some of the proteins or DNA—to the cover slip, then you can actually image two individual molecules interacting with each other, basically that’s what we do.”
What does that look like?
“We get a lot of pictures where it’s a lot of little spots that looks kind of like astronomical images, and then we can do some analysis to see where are those spots,” she said. “How bright are those spots? If we’re doing two-color imaging, do two colors co-localize? Basically, I try to use this technique to explore physically what’s happening when two molecules interact.”
Etson’s research falls into a new and exciting area of research that utilizes single-molecule techniques—that is, techniques that allow observation of just one of each molecule of interest. This is different than many more commonly used techniques, which observe a whole population, or ensemble, of molecules at once. Why is it important to observe molecular interactions on the single molecule level? Is there any reason why one must go through all these delicate protocols and use ultra-precise technologies instead of say, observing a vat of DNA and proteins?
“There are a few reasons why you might want to do things at the single molecule level,” Etson said. “So, first off, if you’re trying to look at either a binding reaction or the production of some kind of product, you need to have some way to have all of the partners in the reaction begin at the same time. If you’re doing an ensemble experiment, you need some kind of way to synchronize.”
Dr. Etson explained that this synchronization can be achieved in many instances.
“But then what you’re getting is sort of an average of how long it takes this large ensemble of molecules to undergo this process,” she said. “And that means that if there’s some heterogeneity among those molecules, you won’t see it, because all you’ll ever [observe] is the average.” Single-molecule experiments, such as those performed in the Etson lab, can help to parse out this heterogeneity, and provide a more complete picture of what is happening at the molecular level than ensemble experiments can.
“There are two different types of heterogeneity you can get,” she said. “You can have static heterogeneity, which is differences among individual molecules, which can be interesting. Or you can have dynamic heterogeneity, which is each molecule changes over time, and that can be also important. There’s a lot of work that’s gone into understanding exactly how proteins mediate reactions.”
Etson continued on to explain this heterogeneity in more detail.
“The idea that fits in with this heterogeneity is that if the molecule can explore different states—meaning parts of the molecule maybe shift a little bit—that might mean that sometimes the molecule is highly active and sometimes it’s not highly active,” she said. “Then the same question could go into static heterogeneity; maybe some of [the single molecules] are really super active and some of them are not as active. So understanding what is going on with that—what are the causes of heterogeneity? how much of it is there?—can be really important just for understanding how things work.”
At the moment, Dr. Etson is investigating a class of proteins called restriction endonucleases, or restriction enzymes. These are well-known proteins often used in genetic engineering for their ability to cut through a strand of DNA when they recognize a specific pattern in the DNA sequence. While often used, there is still some mystery as to their evolution simply waiting to be brought to light. This is only one reason, however, that this class was chosen to be Dr. Etson’s system of study.
“The other reason I chose them is that I feel like they’re a very good model system for trying to study both protein-DNA interactions and catalysis, because they interact with DNA in multiple ways,” she said. “They’re known to have specific and nonspecific interactions with the DNA. They also bend DNA in some cases upon binding, and then they cleave DNA. So you have lots of different processes that you can study using this one class of enzyme.”
Etson elaborated on her long-term goals.
“My ultimate goal is to try to learn something about these enzymes, but also develop some tools that could be adapted to other types of proteins,” she said. “Right now, I’m not studying anything that’s going to cure cancer, but obviously cancer is a process that involves DNA and proteins, so there could be important targets that you could use [a technique] I’ve developed to study.”
Etson’s research does not simply study the interactions between protein and DNA. In reality, her lab works to develop novel insights into how scientists look at protein-DNA interactions. In essence, Etson’s research seeks to provide a new, unabridged look into how these ever-important molecules are pulling our strings.
“Understanding in a deeper way how proteins interact with DNA is extremely important,” she said. “And in particular, single-molecule techniques have made it possible for us to observe that although a lot of proteins have specific binding sites on DNA, they most likely interact with DNA in a lot of other ways nonspecifically, and that those interactions can be really important for understanding how they do the job that you’re most interested in, which might involve the specific binding site.”