Green Fluorescent RNA Helps Reveal the Inner Workings of Living Cells

Scientists have been using fluorescent proteins, ultraviolet light and optical microscopes since the 1990s to tag and observe living cells. This groundbreaking technique, based on a naturally occurring protein found in bioluminescent jellyfish, garnered a Nobel Prize in Chemistry.

Subsequent research has focused not only on improving protein tagging and tracking but also developing similar methods for studying the role of RNA in cellular activity.

One such method uses “Spinach,” a synthetic fluorescent molecule assembled in 2011 by researchers at New York’s Weill Cornell Medical College, led by pharmacology professor Samie Jaffrey, M.D., Ph.D.

Recently, Jaffrey’s group teamed up with scientists at the National Heart, Lung, and Blood Institute and the UK Medical Research Council to map and miniaturize “Spinach,” providing a basis for better understanding the inner workings of living cells. Their findings appear in the August 2014 issue of Nature Structural & Molecular Biology.

A key challenge in observing proteins or RNA inside cells is knowing whether the fluorescent tags are influencing intracellular activity: the smaller the tag, the lesser the likelihood of interactions that might affect normal cell function. For this reason, the team sought to produce what they aptly call “Baby Spinach”; a fluorescent RNA tag about half the size of the original.

“After we developed 'Spinach,' creating a smaller version was always our goal,” Jaffrey said. “When we use fluorescent tagging there is always a risk that the tag will change the structure of the RNA we want to study. Because 'Baby Spinach' is smaller, we now can introduce small tags that have minimal perturbing effects.”

Team members were able to design the smaller tag only after learning more about the structure of the original “Spinach.”

“We developed a detailed blueprint of this fluorescent RNA molecule,” said Adrian Ferré-D'Amaré, M.D., Ph.D., who heads the NHLBI Laboratory of RNA Biophysics and Cellular Physiology. “And now we have a foundation for designing or improving other fluorescent RNAs.”

Major support for their work was provided by the NHLBI’s intramural research program. Additional NIH support included research and training grants from the National Institute of General Medical Sciences and National Institute of Neurological Disorders and Stroke (NINDS).

The work to develop “Spinach” at Weill Cornell Medical College was reported in the July 29, 2011, issue of Science. That research was funded in part by the NINDS as well as a training grant from the National Cancer Institute.

Students Lead RNA Research 

Two of the researchers that developed “Baby Spinach” are National Institutes of Health Oxford-Cambridge Scholars, participating in an accelerated doctoral training program for outstanding science students committed to biomedical research careers.

Katherine Warner, principal author of the “Baby Spinach” article, and co-author Michael Chen are working with research mentors at the NHLBI and University of Cambridge on collaborative doctoral projects. Students in the program earn a PhD from either Oxford or Cambridge universities in the U.K., in about half the time it takes to complete a biomedical Ph.D. program in the United States.

Warner said the NIH Oxford-Cambridge Scholars Program has jump-started her research career.

“During the past four years, I’ve designed experiments for ground-breaking studies and developed international collaborations with experts in chemistry, biophysics, and structural and plant biology,” she said. “The program has allowed me to hone my skills as a scientist and develop my sense of independence.”

The program fully funds tuition and stipend support for United States citizens and permanent residents for the duration of their Ph.D. training.

To learn more about the NIH Oxford-Cambridge Scholars Program, contact Matt Vogt, Director of Student Affairs; email vogtm@mail.nih.gov or tel. 301-435-1513.

Why Study RNA Structure? 

RNA mediates the flow of genetic information in all living things, determining which genes contained in DNA are translated into functional proteins.

Malfunctioning RNA is associated with many human diseases, including ALS (amyotrophic lateral sclerosis), cancer, muscular dystrophy, and retinitis pigmentosa. Moreover, some viruses, such as Ebola hemorrhagic fever, influenza and measles, store their genetic information in RNA instead of DNA.

Finding treatments for such genetic and viral diseases may depend on understanding the structure of their RNA, which folds into a variety of intricate shapes that determine how it functions.

Want to learn more about RNA folding? Try playing EteRNA, a free Web-based game created by researchers at Carnegie Mellon and Stanford universities with funding from the National Institute of General Medical Sciences.

In the game, players design their own RNA molecules. Stanford biochemists then build the most promising player-designed molecules in the lab, both to see if they fold into stable, predictable structures and to discover more about how RNA folding works. The researchers believe this work will eventually lay a foundation for developing new RNA-based therapies for disease.

Read more about how RNA function affects human health in the NIGMS publication The New Genetics.