The
ability to tag proteins with a green fluorescent light to watch how
they behave inside cells so revolutionized the understanding of protein
biology that it earned the scientific teams who developed the technique
Nobel Prizes in 2008. Now, researchers at Weill Cornell Medical College
have developed a similar fluorescent tool that can track the mysterious
workings of the various forms of cellular RNA.
In
the July 29 issue of Science, the Weill Cornell investigators report
how they developed an RNA mimic of green fluorescent protein (GFP)—which
they dubbed Spinach—and describe how it will help unlock the secrets of
the complex ways that RNA sustains human life as well as contributes to
disease.
“These
fluorescent RNAs offer us a tool that will be critical for
understanding the diverse roles that RNA plays in human biology,” says
the study’s senior author, Dr. Samie Jaffrey, an associate professor of
pharmacology at Weill Cornell Medical College.
In recent years, the many roles played by RNA have become clearer.
“Scientists
used to think that RNA’s function was limited to making proteins and
that these proteins alone dictated everything that happened in cells,”
he says. “But now we are understanding that cells contain many different
forms of RNA—and some RNAs influence cell signaling and gene expression
without ever being used for synthesizing proteins.”
The
list of known types of RNA has grown rapidly over the past several
years—from messenger RNA that codes for proteins, to diverse
“non-coding” RNAs that affect translation and gene expression, and in
some cases bind to proteins and regulate their function—yet little is
known about how these RNAs work, the researchers say.
The
study’s first author, Dr. Jeremy Paige, who conducted the research as a
graduate student in pharmacology at Weill Cornell Medical College, adds
that the new technology may provide insights into the development of
common disorders. “More and more diseases are being linked to
misregulation of RNA, but without being able to see the RNA, we can’t
understand how these processes lead to disease.
“We hope our RNA mimics of GFP open up the road to discovery,” he says.
The
RNAs developed by the Jaffrey group function like GFP, a natural
protein expressed in jellyfish that exhibits a green fluorescence. GFP
has enabled scientists to watch how proteins move in cells, providing
powerful new insights into their roles in cell function. The DNA that
encodes GFP is placed next to a gene that encodes for a protein,
resulting in the expression of a protein fused to GFP, which can be
observed by specialized forms of microscopy.
To
make an RNA that functions like GFP, the Weill Cornell investigators
took advantage of the ability of RNA to fold into complex
three-dimensional shapes. Their goal was to create two new entities: a
synthetic RNA sequence that would adopt a specific shape, and a small
molecule that would bind to the new RNA and begin to fluoresce.
“These
were two huge challenges,” says Jaffrey. “One challenge was to come up
with an RNA sequence that could ‘switch on’ a small molecule. The other
big hurdle was to find a small molecule that would fluoresce only when
we wanted it to and would not be toxic to cells.”
They
tried a number of molecules, most of which stuck to oily lipids in the
cell membrane and started fluorescing, or they would kill the cell.
Finally, the team realized that GFP itself had a molecule, a
fluorophore, within it that switched its light on when it was bound in a
certain way within the protein. They created chemical molecules based
on the shape of this fluorophore and then developed an artificial RNA
sequence, or “aptamer,” that held the fluorophore in exactly the same
way that GFP held its fluorophore. They named this RNA “Spinach” for its
bright green fluorescence.
The
researchers went even further. They also developed several other
RNA-fluorophore pairs, in addition to Spinach, that each emit a
different fluorescent color, just as GFP has been evolved to exhibit a
palette of colors that helps researchers track many proteins at once.
Whereas GFP derivatives are often named after fruits, the Weill Cornell
researchers named their RNA mimics of GFP after vegetables — Spinach,
Carrot and Radish.
The
Weill Cornell investigators have already begun to use Spinach to track
non-coding RNAs in cells. “Our laboratory has been very interested
understanding why defects in RNA trafficking and translocation lead to
developmental disorders in children, such as mental retardation,” says
Jaffrey. Using Spinach, they were able to watch as a non-coding RNA,
fluorescing green, rapidly clusters in response to cellular stress. “We
expect that Spinach will provide new insights into RNA trafficking in
cells, and how this is affected in medical disorders,” he says.
“There
is still a lot of mystery surrounding RNA in biology. Fluorescent
labeling and imaging has proved to be a powerful tool for scientists in
the past, and we are hoping that Spinach too will be a tool that helps
accelerate scientific discovery,” says Paige.
Dr. Karen Wu of the Department of Pharmacology is a co-author on the study.
The
work was supported by the McKnight Neuroscience Technology Innovation
Award and the National Institutes of Neurological Disorders and Stroke.
Weill Cornell Medical College has filed a patent application on the technology.
