The code breaker, p.6
The Code Breaker, page 6
The field of biochemistry provided many answers by showing how the chemical molecules in living cells behave. But there was a specialty that looked even deeper into nature: structural biology. Wielding imaging techniques such as X-ray crystallography, which is what Rosalind Franklin used to find evidence of the structure of DNA, structural biologists try to discover the three-dimensional shape of molecules. Linus Pauling worked out the spiral structure of proteins in the early 1950s, which was followed by Watson and Crick’s paper on the double-helix structure of DNA.
Doudna realized that she would need to learn more about structural biology if she wanted to truly understand how some RNA molecules could reproduce themselves. “To figure out how these RNA do chemistry,” she says, “I needed to know what they looked like.” Specifically, she needed to figure out the folds and twists of the three-dimensional structure of self-splicing RNA. She was aware that such work would be an echo of that done by Franklin on DNA, and the parallel pleased her. “She had a similar kind of question about the chemical structure of a molecule that was at the heart of all of life,” Doudna says. “She believed that its structure would provide all sorts of insights.”1
Rising star at Yale
Doudna also sensed that once you figured out the structure of a ribozyme, it might lead to groundbreaking genetic technologies. The citation for the Nobel Prize that Thomas Cech won with Sidney Altman hinted at what this might be: “A futurist possibility is to correct certain genetic disorders. Such a future use of gene shears will require that we learn more about the molecular mechanisms.” Gene shears. Yes, the Nobel committee was prescient.
This pursuit meant that it was time to move on from the lab of Jack Szostak, who admitted to not being a visual thinker or expert in structural biology. So in 1991, Doudna considered where she could do her postdoctoral work. There was one obvious choice, the structural biologist who had just shared the Nobel Prize for discovering the catalytic RNA that she and Szostak had been studying: Thomas Cech (pronounced “check”) of the University of Colorado in Boulder, who was using X-ray crystallography in order to explore each nook and cranny of the structure of RNA.
Thomas Cech
Doudna already knew Cech. He was the one who whispered “Good job” after her sweaty-palmed lecture at Cold Spring Harbor in the summer of 1987. She had met him again when she took a trip to Colorado that year. “Because we were sort of friendly competitors both racing to make discoveries about the self-splicing introns, I sent him a note,” she recalled.
It was a real note, on paper, because email was not yet common. She wrote that she was going to be traveling through Boulder and asked if it would be possible to visit his lab. To her surprise, he quickly got back to her, telephoning one day when she was at work in Szostak’s lab. “Hey, Tom Cech is on the phone for you,” the colleague who picked up the phone called out. Her lab mates gave her a curious look, but she just shrugged.
They met in Boulder on a Saturday. Cech had brought his two-year-old daughter to the lab, and he bounced her on his knee as he talked to Doudna, who was completely charmed by both his mind and his fatherly instincts. Their encounter was an example of the mix of competition and collegiality that marks scientific research (and many other endeavors). “I think the reason Tom met with me was that the Szostak Lab was doing work that was potentially competitive but also that there might be opportunities to learn from each other,” she says. “And he probably thought it was a way to get some information about what our lab was up to.”
After she earned her PhD in 1989, she decided to do her postdoctoral work with Cech. “I realized that if I really wanted to figure out the structure of RNA molecules, my smart move was to go to the very best RNA biochemistry lab,” she says. “Who can be better than Tom Cech? This was the lab that had first discovered self-splicing introns.”
Tom Griffin
There was one other reason that Doudna decided to go to Boulder for her postdoctoral work. In January 1988 she had married a Harvard Medical School student named Tom Griffin, who was working in a lab next to hers. “He saw in me things I didn’t see at the time, including capabilities in science,” she says. “He pushed me to be bolder than I would have been.”
Griffin, from a military family, loved Colorado. “When we were thinking of where to go when we finished our degrees, he really, really wanted to move to Boulder,” says Doudna. “I realized that if we went to Boulder, I could work with Tom Cech.” So they moved there in the summer of 1991, and Griffin got a job at a startup biotech company.
At first the marriage worked well enough. Doudna bought a mountain bike, and they would ride along Boulder Creek. She also took up roller-blading and cross-country skiing. But her passion was science, and Griffin didn’t have her single-minded focus. Science for him was a nine-to-five endeavor, and he had no aspirations to be an academic researcher. He loved music and books, and he became an early fan of personal computers. Doudna respected his broad range of interests but didn’t share them. “I’m someone who’s thinking about science all the time,” she says. “I’m always focused on what’s cooking in the lab, the next experiment, or the bigger question to pursue.”
Doudna believes their differences “say something negative about me,” though I’m not sure she really believes that, nor do I. People are different in their approaches to their work and passions. She wanted to spend weekends and nights in the lab doing experiments. Not everybody should be that way. But some people should.
After a few years, they decided to go their separate ways and get a divorce. “I was obsessed with what my next experiment was going to be,” she says. “He didn’t have that same intensity. That just created a critical wedge that was not fixable.”
The structure of a ribozyme
Doudna’s mission when she arrived at the University of Colorado as a postdoc was to map the intron that Cech had discovered could be a self-splicing piece of RNA, showing all of its atoms, bonds, and shapes. If she succeeded in figuring out its three-dimensional structure, that would help show how its twists and folds could bring the right atoms together to cause chemical reactions and allow the snippet of RNA to replicate itself.
It was a high-risk venture, one that involved going to a region of the playing field where few others wanted to run. At the time there was not much work being done on RNA crystallography, and most people would look at her like she was nuts. But if she succeeded, there would be a huge payoff for science.
During the 1970s, biologists had figured out the structure of a smaller and simpler RNA molecule. But little progress had been made in the twenty years since then because scientists found it difficult to isolate and get images of bigger RNAs. Colleagues told Doudna that getting a good image of a large RNA molecule would, at that time, be a fool’s errand. As Cech put it, “If we had asked the National Institutes of Health to fund this project, we would have been laughed out of the room.”2
The first step was to crystallize the RNA—in other words, convert the liquid RNA molecule into a well-organized solid structure. That was necessary in order to use X-ray crystallography and other imaging techniques to discern its components and shapes.
Helping her was a quiet but cheery graduate student named Jamie Cate. He had been using X-ray crystallography to study the structure of proteins, but when he met Doudna he joined her quest to focus on RNA. “I told him about the project I was working on and he got very interested,” she says. “It was really out there. We had no idea what we were going to find.” They were pioneering a new field. It was not even clear that RNA molecules would have well-defined structures like proteins do. Unlike Tom Griffin, Cate loved to focus on lab work. He and Doudna would talk every day about how to crystallize the RNA, and soon they were continuing their discussions over coffee and sometimes dinner.
One breakthrough came as a result of the random things that often happen in science: a slight blunder, like the mold that got on Alexander Fleming’s Petri dishes and led to the discovery of penicillin. One day a technician was working with Doudna to try to make crystals, and she put the experiment into an incubator that was not working properly. They thought the experiment was spoiled, but when they looked at the samples through a microscope they could see crystals growing. “The crystals had RNA in them and were beautiful,” Doudna recalled, “and that was the first breakthrough showing us that to get these crystals we had to elevate the temperature.”
Another advance shows the enduring power of being in the same location as other smart people. Tom and Joan Steitz, a husband-and-wife team of Yale biochemists who were studying RNA, were on sabbatical in Boulder for a year. Tom was particularly sociable and liked hanging around the lunchroom of the Cech lab holding a mug of coffee. Doudna mentioned to him one morning that she had been able to get good crystals of the RNA molecule she was researching, but they tended to break down too quickly when they were exposed to X-rays.
Steitz replied that in his Yale lab he had been testing a new technique for cryocooling crystals. They plunged crystals into liquid nitrogen so they would freeze very rapidly. That helped to preserve the structure in the crystals even when they were exposed to X-rays. He arranged for Doudna to fly to Yale and spend time with the researchers in his lab there who were pioneering the technique. It worked beautifully. “At that point we knew that we had crystals that were ordered enough that we would eventually be able to solve the structure,” she says.
Yale
Her visit to Tom Steitz’s lab at Yale, where innovative techniques and equipment such as cryocoolers were being funded, helped convince Doudna to accept a job there in the fall of 1993 as a tenure-track professor. Not surprisingly, Jamie Cate wanted to accompany her. She contacted the Yale authorities and helped arrange for him to transfer there as a graduate student in her lab. “They required him to retake his qualifying exams,” she says, “and as I’m sure you can imagine, he aced them with flying colors.”
By using the super-cooling techniques, Doudna and Cate were able to create crystals that diffracted X-rays well. But they were stymied by what is known in crystallography as the “phase problem.” X-ray detectors can measure properly only the intensity of a wave but not the phase part of the wave. One way to attack the problem is to introduce a metal ion into a few regions of the crystal. The X-ray diffraction pictures show the position of the metal ions, and that can be used to help calculate the rest of the molecular structure. That had been done with protein molecules, but no one had figured out how to do it with RNA.
Cate solved the problem. He did it by using a molecule called osmium hexamine, which has an interesting structure that lends itself to interacting in a few nooks of RNA molecules. As a result, the X-ray diffractions could produce an electron-density map that would provide clues for the structure of an important folded region of the RNA they were studying. They began the process of creating these density maps and then building models of potential structures, just as Watson and Crick had done for DNA.
Her father’s farewell
When their work was reaching its climax in the fall of 1995, Doudna got a call from her father. He had been diagnosed with melanoma, and it had metastasized to his brain. He told her that he had only three months to live.
She spent the rest of that fall flying back and forth from New Haven to Hilo, a journey of more than twelve hours. Chunks of time spent at her father’s bedside were interspersed with hours on the phone with Cate. Each day, Cate would send her a new electron-density map by fax or email, and they would talk about ways to interpret it. “It was an incredible time of highs and lows and intense emotional swings,” she recalls.
Fortunately, her father was genuinely curious about her work, and that made the ordeal less painful. In between periods of pain, he would ask her to explain the latest images she had received. She would walk into his bedroom, and he would be lying there looking at the latest data. Before they could discuss his health, he would begin asking questions. “It would remind me of his scientific curiosity and how he had shared it with me when I was a child,” she says.
During a visit that November, which lasted through Thanksgiving, an electron-density map arrived from New Haven that she realized was good enough to nail down the structure of the RNA molecule. She could actually see how the RNA was folded up into an amazing three-dimensional shape. She and Cate had been working on it for more than two years, while countless colleagues had declared what they were doing was impossible, and now the latest data showed that they had triumphed.
Her father was completely bedridden by then and could barely move. But he was lucid. She walked into his bedroom and showed him a color printout she had made from a data file of the latest map. It looked like a green ribbon that was twisted into a really cool shape. “It looks like green fettuccini,” he joked. Then he got serious. “What does it mean?” he asked.
By trying to explain it to him, she was able to clarify her own ideas about what the data meant. They pored over a region on the map that was caused by a cluster of metal ions, and she speculated on the ways that the RNA could be folding around such a cluster. “Maybe there’s a core of metals here that helps this RNA to fold up into this type of twist,” she suggested.
“Why would that be important?” he asked. She explained that RNA is made up of very few chemicals, so it accomplishes complex tasks based on the different ways it is folded. One of the challenges with RNA is that it’s a molecule made of only four chemical building blocks, unlike proteins, which have twenty. “Because there is a lot less chemical complexity to RNA,” she says, “the challenge is to think about how does it fold into a unique shape.”
The visit clarified how time had deepened her relationship with her father. He took science seriously, and he took her seriously. He was attracted to all the details, but he also sought the bigger picture. She recalled the times she had visited his classroom and seen his excitement at communicating his passions. She also recalled, less happily, the times that she had gotten angry at him because she thought he made snap judgments, some of them prejudiced, about people. Bonds can take different forms, both in chemistry and in life. Sometimes an intellectual bond is the strongest.
* * *
When Martin Doudna died a few months later, Jennifer and her mother and sisters went with friends on a hike to scatter his ashes high up in the Waipio Valley near Hilo. The name means “curved water,” and the river that winds through its lush wilderness has many gorgeous waterfalls. Among those joining them were Don Hemmes, the biology professor who mentored Jennifer, and her closest childhood friend, Lisa Hinkley Twigg-Smith. “As we released his ashes into the wind,” Twigg-Smith recalled, “an endemic hawk known as an ‘io, which is associated with the gods, soared overhead.”3
“It was only after he died that I realized how influential he was in my decision to become a scientist,” Doudna says. Among the many gifts that he gave her was a love of the humanities and how it intersects with the sciences. The need for that was becoming clearer to her as research led her into realms that required moral guideposts as well as electron-density maps. “I think my father would have loved to understand CRISPR,” Doudna reflected. “He was a humanist, a humanities professor, who also loved science. When I talk about CRISPR’s effects on our society, I can hear my father’s voice in my head.”
Triumph
Her father’s death coincided with her first major scientific success. She and Cate, along with their lab colleagues, were able to determine the location of every atom in a self-splicing RNA molecule. Specifically, they showed how the structure of a key domain of the molecule allowed RNA to pack helices together to create its three-dimensional shape. A cluster of metal ions in that domain formed a core around which the structure folded. Just as the double-helix structure of DNA revealed how it could store and transmit genetic information, the structure discovered by Doudna and her team explained how the RNA could be an enzyme and was able to slice, splice, and replicate itself.4
When their paper was published, Yale sent out a press release that attracted the notice of a local New Haven television station. After trying to explain what a ribozyme is, the news anchor reported that it had baffled scientists because they had never been able to see its shape. “But now a team led by Yale scientist Jennifer Doudna finally was able to capture a snapshot of the molecule,” the anchor proclaimed. The story featured a young, dark-haired Doudna in her lab, showing off a blurry image on her computer screen. “We hope our discovery will provide clues as to how we might be able to modify the ribozyme so that it can repair defective genes,” she said. It was a momentous statement, though she didn’t think about it much at the time. It would be the beginning of a quest to translate basic science about RNA into a tool that could edit genes.
In another, more sophisticated television report, done by a syndicated science news show, Doudna appeared in a white lab coat using a pipette to put a solution into a test tube. “It’s been known for fifteen years that RNA molecules could function like proteins in cells, but nobody knew how that could be, because nobody has really known what RNA molecules look like,” she explained. “We have now been able to see how an RNA molecule can form itself into a complicated three-dimensional structure.” Asked what the implications could be, she again pointed to what would be her future work: “One possibility is that we might be able to cure or treat people who have genetic defects.”5
Over the next two decades, many people would contribute to the development of gene-editing technologies. What distinguishes Doudna’s tale is that, by the time she entered the field of gene editing, she had already established her reputation and earned distinction in the most basic underlying science: the structure of RNA.
