The rockefeller university

Jennifer Doudna’s stubborn streak has served her well. Early in her career, she wanted to understand how RNA molecules perform catalytic feats long thought to be the bailiwick of proteins. Dogma held that RNAs would not yield to the standard technique by which scientists peer into the workings of complex molecules. Nonetheless, Doudna latched onto this venture and produced the first detailed portrait of a large RNA. She has consistently applied her perseverance and ingenuity to illuminate novel aspects of RNA structure and function, many of which were unimagined when she first stepped into the lab.

As a child in Hilo, Hawaii, Doudna wanted to make a difference in the world, but she couldn’t imagine how that was going to happen. She was acutely aware that she lived on a volcanic rock in the ocean. Events she read about in the newspaper occurred far away. “Even popular music groups didn’t visit my town or my state,” Doudna says.

Doudna’s parents were teachers, and her literature professor father, especially, cultivated an intellectual atmosphere at home. His interests ranged widely, and they included nature and the environment. Over dinner, he tried to pique his daughter’s curiosity about Hawaii’s natural resources or topics he’d encountered by reading Lewis Thomas and John McPhee. Years later, he sent Doudna books and wrote letters about who we are as humans, why we are here, and what makes us special. On visits while she was a postdoctoral fellow, he grilled her about her experiments and why they mattered—not to test her, but because he hungered to know and held Doudna and her passions in high regard.

When Doudna was young, she loved mathematics and puzzles. Science bored her, as she viewed it through the lens of textbooks that presented a “dry bunch of facts to memorize for a test,” she says. At around age 12, she read The Double Helix, James Watson’s description of the race to elucidate the structure of DNA. The book revealed an enterprise rich in people’s foibles, flaws, and occasional strokes of brilliance. It opened her to the world of research as it is practiced. Although the enterprise’s human aspects appealed to her, she was scarred by the dull presentation in school. Finally, in 10th grade, Doudna’s chemistry teacher broke through. Miss Wong explained that science is about asking questions and figuring out how to answer them. Doudna was intrigued, but the only female scientist she knew about was Marie Curie, who was dead.

In the meantime, her dad had begun taking her on walks with friend, biologist, and mushroom expert Don Hemmes. They explored lava tubes and other dark, moist habitats, admiring the diverse colors and shapes of the fungi, many of which had never been categorized. The trio took photos of their finds and later tried to identify them. This experience exposed Doudna to the joy of discovery. By the time she entered Pomona College, she had decided to pursue biochemistry.

In graduate school at Harvard, Doudna heard that faculty member Jack Szostak was exploring the possibility that RNA molecules lay at the origin of life. Like its DNA cousin, RNA holds genetic information. Scientists had discovered several years earlier that RNA can catalyze chemical reactions, some of which resemble those that synthesize new RNA. This observation raised a provocative possibility. The capacity to reproduce is an essential property of living things, and that process requires an organism to duplicate its genetic material. A self-replicating RNA might have materialized in the primordial soup and led to the first cell. Szostak was probing the feasibility of that idea.

Undeterred, she chose to discuss a paper that describes how the antibiotic penicillin inhibits its target enzyme when she gave a required seminar. That study “profoundly affected” her scientific trajectory, she says. To Stubbe, “it was a revolution that you could take your understanding of chemical structure to understand how an essential bacterial enzyme was inactivated,” she says. The allure of unraveling an enzyme mechanism at the atomic level propelled much of her subsequent inquiry.

Drawn by his vision and energy, and awed by what she regarded as his genius, Doudna joined Szostak’s lab. They aimed to engineer a catalytic RNA—an RNA enzyme or “ribozyme”—that would produce copies of itself. To do so, it would need to use a second RNA molecule as a template, a property not seen in natural molecules, which rely on internal sequences to do their jobs. The team used numerous tricks, including splitting the original RNA into several components, to create a ribozyme that could stitch together short pieces of RNA that matched external templates. This success lay the groundwork for a tremendous amount of subsequent work by Doudna and others in the Szostak lab. It culminated, several years later, in a ribozyme that could duplicate its own entire sequence.

One day during Doudna’s trial period in Szostak’s lab, he sought her out to pick her brain. “I was blown away,” she says. He was a tenured Harvard faculty member whom she venerated, yet he valued her ideas. “Later when I had to make decisions on my own, I remembered that conversation,” she says. “If Jack thought my opinion mattered, it must be worth something.”

Doudna’s confidence, presumably sparked by innate wiring, kindled by respectful parenting and inspiring grade school teachers, and stoked by Szostak’s recognition of her keen mind, has buoyed her through challenging times. One of these came soon after graduate school. By then, Doudna was wondering how catalytic RNAs work. To understand the mechanisms, she needed to know their structures—how their chemical groups are positioned so they can bind substrates, attack atoms, and thus accelerate reaction speeds.

Scientists had deciphered some of the principles that influence protein folding, but analogous guidelines did not exist for RNAs. How these molecules assume distinct shapes that perform the types of reactions previously associated with proteins was mysterious. Among other conundrums, only four closely related subunits make up RNAs, whereas twenty subunits, with varied chemical repertoires, compose proteins.

To determine the structure of a complicated molecule, researchers often crystallize it and then shoot X-rays at it. The resulting scatter off the atoms produces a pattern that reveals the molecule’s architecture. The method requires that the target packs into a stable three-dimensional configuration, and conventional wisdom suggested that RNAs would not. They were too floppy, scientists thought, and RNA’s backbone carries many negative charges, which repel one another and would impede compact folding. In addition to these and other theoretical barriers, X-ray crystallography had been applied successfully to only one RNA before, despite much effort over a couple of decades. Many investigators had concluded that the single achievement had been a fluke.

“Plenty of people said it was crazy and would never work,” Doudna says. But if RNAs had sparked life, their 3D structures had forged a crucial part of the story. Despite the poor prospects, she decided to tackle the problem. She joined the lab of Thomas Cech at the University of Colorado as a postdoctoral fellow and aimed to crystallize a crucial portion of a particular ribozyme’s active site.

Two years and many failed attempts later, she and her colleagues decided to try a new method that involved quick cooling crystals in liquid nitrogen. After numerous tries, they put yet another sample onto a tiny loop—essentially a miniature soup ladle—made of fishing line. Then, like dipping an egg into a glass of dye, they lowered it into a vial of liquid nitrogen and mounted the superfrozen crystal on an X-ray diffraction device.

As usual, the detector revealed a circular pattern of spots. The farther it reaches, the higher the image’s resolution. Until this experiment, the data always clustered in the middle of the screen.

This time, it touched the edge.

“I was ecstatic,” says Doudna. “It was a moment of pure joy.” All the effort she and her colleagues had invested over the past several years would pan out. They would eventually be able to calculate the structure.

Doudna set up her own lab at Yale in 1994 and finished the analysis. The interactions the team uncovered showed not only how this particular RNA folds up, but revealed the principles that hold together RNAs in numerous biologically important RNA-protein machines.

Her investigations at Yale and subsequently at the University of California, Berkeley, where she moved in 2002, have extended far beyond ribozymes. She has studied, for instance, how the hepatitis C virus hijacks the protein-manufacturing apparatus of mammalian cells and how a particular enzyme, Dicer, generates small RNAs that destroy target messenger RNAs and thus thwart the production of certain proteins.

Doudna’s expertise about RNA structure and RNA-protein interactions prepared her to think creatively about the CRISPR/Cas9 system that has drawn fame for its gene-editing potential. Doudna’s work in this realm holds the potential to repair flawed genes or insert new ones into a wide variety of cells, including those of plants and humans.

Doudna’s dedication and courage have fueled a bold and exceptional research program. “You always encounter difficulties and challenges,” she says. “You have to believe in what you’re doing to get you through those times.”

Author: Evelyn Strauss, Ph.D.