Scientists have long fought cancer with their biggest, baddest research tactics — but big and bad only go so far. A new, potential approach involves working in a more concealed space: within the ends of our chromosomes. Biochemist Michael Stone, an assistant professor at the University of California, Santa Cruz, and his colleagues are tackling cancer research with, literally, a twist of biophysics. Stone’s lab probes inside the tips of chromosomes to stretch and bend DNA, watching how each strand reacts to invasive prodding.
Warping DNA requires specific and precise instruments. These adjustments in structure are so minuscule, less than one billionth of a meter, that only special microscopes can detect the changes. Stone’s lab is applying a pair of novel techniques — fluorescence microscopy and “magnetic tweezers” — to contort DNA precisely and, at the same time, account for each infinitesimal change in structure.
The magnetic tweezers and fluorescence microscopy duo are stirring new ideas about how folded structures in our cells may serve as the next major drug target for cancer therapies.
“We have this unique niche where we’re not only up on the biology, but we also have these specialized techniques,” Stone says. “We study how our genetic material behaves as just that, a material. That’s not the way we’re typically taught to think about DNA.”
The cells in our bodies carry a genetic blueprint tucked into compact pieces of DNA called chromosomes. A cap at the end of each chromosome protects the DNA from being damaged. These protective ends, called telomeres, play a key role in differentiating between chromosomes that end naturally with telomeres, and chromosomes that have damaged ends without telomeres. Scaled to fit in your hand, a chromosome would appear much like a shoelace, with the telomere acting as the plastic tip that keeps the lace from unraveling.
Every time our cells divide and copy, each telomere loses a bit of its length. Shortening telomeres essentially act as an hourglass for the life span of the cell. The chromosome divides and shrinks until it becomes a health risk to the cell, at which point, it enters a death phase. Though “death phase” sounds fairly ominous, it’s actually what’s keeping our cells in check. Every cell division is an opportunity for chromosomal mutation. The cell’s limited lifespan, caused by telomere shortening, restricts mutation and genetic damage.
Not all cells divide at the same rate though. Some cells—like stem cells—need to divide more rapidly to successfully function. In these kinds of cells, an enzyme called telomerase makes new telomeric DNA, enabling the rapid divisions by keeping the ends healthy. However, the quick and numerous cell divisions telomerase facilitates can also play a darker role in human health.
Over years of cancer research, scientists have repeatedly come to the same conclusion: telomerase is a fundamental contributor in creating most cancerous cells. The growth of a cancer cell is uncontrolled and its lifespan, virtually immortal. Research has found overactive telomerase in 90% of cancer cells, perpetuating cell growth that develops into tumors.
By tweaking the telomeres’ structure, Stone’s lab at UCSC reaches outside the usual realm of techniques used to study DNA. “My lab is one of the few groups in the world digging deep biophysically into the molecular mechanisms of telomere length maintenance, which is very important in medical biology,” he says.
The magnetic tweezers and a fluorescence microscopy technique called Förster Resonance Energy Transfer (FRET) may look like a convoluted duo to the untrained eye. But the basic ideas are simple: the “tweezers” pull and twist on molecules of DNA, while FRET measures the distances in real time.
The instruments reside in a small, busy lab room. At first sight it’s overwhelming, but it’s a purposeful clutter. What appears to be a haphazard maze of varying sizes of glass is a painstakingly aligned series of laser-guiding lenses and mirrors. Correctly aligned, the optics direct the laser that powers FRET. In other words, it’s the kind of display best kept at arm’s length.
Near the tweezers, a computer screen displays images of the manipulated DNA. With one end of the DNA molecule secured to a glass plate, scientists attach a magnetic bead to the other end and place a larger magnet above the bead. As the large magnet moves closer to the bead, the force on the DNA increases, stretching it further. During this process, FRET measures the fractional changes in length within single molecules by monitoring the interactions between a green donor FRET dye and a red acceptor FRET dye. The amount of energy transfer between the two dyes directly corresponds to the distance between them, making it one of the world’s smallest measuring tapes.
“Basically our lab married these two techniques so we could apply a very stable force and simultaneously monitor it [the DNA structure] on a very small scale,” says Xi Long, the lead graduate student in Stone’s lab.
The researchers focus on what happens in the telomeres of the chromosomes. They zero in on an unusual structure of DNA known as a G-quadruplex. With a structure just as complicated as its name, the G-quadruplex is no simple query. It fascinates researchers because of its propensity to fold and unfold into different forms, like origami on a molecular level. The effects of these folded forms on the level of telomerase activity are profound. In its folded, stable state, the G-quadruplex inhibits telomerase action, making it an intriguing candidate in the cancer therapy world.
Stone’s lab used the paired magnetic tweezers and FRET technique to explore the role of the G-quadruplex in detail. The tweezers stretch specifically dye-labeled DNA molecules in the structure to reveal the lengths at which folding and unfolding occurs. The team has found that the G-quadruplex is surprisingly touchy. Disturbing a single nucleotide could mean its demise. In that way, it’s a lot like Jenga: tamper with one building block and the whole thing collapses.
With such a temperamental structure, the goal is to find a way to keep the G-quadruplex happily stable and folded. Such a tactic could suggest ways to design a new type of cancer therapy. In the lab, scientists have experimented with stabilizing molecules that keep the G-quadruplex folded, blocking overactive telomerase. Replicating that success in cancerous cells — and, ultimately, in patients — will be a long road, Stone acknowledges.
Stone’s lab is keen to share the skills needed to develop and refine these techniques with other labs and researchers. Keeping them secret would eliminate the opportunity for colleagues to contribute their findings to science on a larger scale, says Stone.
“Now, when we report on it in the literature and at meetings, people studying things different than telomere structure are very excited about the methodology,” says Stone. “There are many applications for this sort of technology, reaching much further than just the interests of my lab.”
On a larger scale, Stone strives to explore how the G-quadruplex influences the broader biological roles of telomeres. For example, the lab will study how replication in telomeres differs compared to other segments of DNA. One salient feature of telomere DNA is its high sequence repetition. Repetitive sequences can lead to challenges in replication by confusing DNA polymerase, the replicating protein. A repeat in DNA causes stalling or back-tracking, like a scratch on a CD makes the music skip or stutter.
Stone and his team have studied the G-quadruplex for more than four years. With such a complex focal point, the lab can be an arduous place to work.
“I tell my younger students that you have to enjoy the process of science to be a good scientist. You can’t be completely results-focused,” says Stone. “Experiments are filled with challenges and difficulties. Taking these challenges in stride is just a part of the daily grind of being a research scientist.”
He gestures to a photo of the New York Yankees stadium pinned to his office wall. “Major league baseball hitters fail way more than they succeed,” Stone says. “In fact, the very best hitters fail seven times out of ten. So if you hit .300, you’re doing really well. You might even be MVP of the league. Yet more times than not, you didn’t get a hit. Science is like that. You need to have resilience.”
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