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Imaging Single Molecules

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Su-A Myong
Title: Assistant Professor, University of Illinois, Urbana-Champaign
Education: PhD, University of
California, Berkeley, 2001
Recommended by: Harris Lewin

Just how the cytosolic protein retinoic acid inducible-gene I detects viral RNA and elicits an antiviral immune response isn't known. Solving the mystery of how it goes about its business is a big focus of Su-A Myong's lab at the University of Illinois, Urbana-Champaign. One of the strongest investigative tools she has is single molecule fluorescence detection. Following up on work from Japanese scientists, who in 2004 discovered the antiviral receptor function of RIG-I, an RNA helicase that can detect replicating double-stranded RNA viruses and subsequently activate immune signaling pathways in the host cell, Myong developed a technique to observe the activity of this protein at the single molecule level.

According to Myong, RIG-I can detect two pathogen-associated molecular patterns, or PAMPs: long, double-stranded RNA with a 5'-triphosphate. Both allow RIG-I to turn on production of type I interferon. "What was not known was if the protein recognized these two in [an] integrated manner, meaning if the two are sensed together, or not," Myong says. But not only is the protein hard to label, it is difficult to detect at the single molecule level using typical fluorescence techniques, such as FRET.

Instead, they came up with protein-induced fluorescence enhancement, or PIFE. Myong's lab discovered that by measuring the intensity changes of a single fluorophore attached to a substrate, they could monitor protein movement on the substrate. "When a protein approaches this fluorophore, the fluorophore becomes much brighter — it seems that it enhances the quantum yield of the fluorophore," she says. With the PIFE assay, a technique they published earlier this year in Science, they were able to monitor the ATPase activity of RIG-I. "[It] turns out that it hydrolyzes ATP and it translocates up and down on [the] double-stranded RNA axis," she says. When they carried out the experiment in the presence of both PAMPs, the translocation activity was about 20 times faster. "In the absence of 5'-triphosphate, the activity of the wild-type protein is very slow, but when you add this triphosphate unit to the RNA, it translocates about 20-fold faster," Myong adds.

Myong credits her start in single molecule imaging to her postdoc mentor — and husband — Taekjip Ha, who is currently the co-director of the Center for the Physics of Living Cells at UIUC. The first project that she and Ha published documented the unexpected repetitive shuttling motion of the E. coli helicase Rep. "Ever since then I was hooked," she says. "It's just amazing the kind of molecular details and the mechanisms of these single proteins that you can measure."

Looking ahead

Myong thinks one of the bigger challenges to the field is finding ways to lend more in vivo relevance to single molecule work. "We face [a] gap between this highly in vitro assay where we use purified protein on a purified substrate and real cells," she says. Five years from now, Myong hopes to see some of the live cell imaging barriers overcome. "The ideal thing would be to monitor single molecules inside a living cell," she adds.

Publications of note

In a 2005 Nature paper, Myong and her colleagues used a single-molecule fluorescence assay to watch the DNA binding and translocation of E. coli Rep, a superfamily 1 DNA helicase homologous to Saccharomyces cerevisiae's Srs2. They found that when Rep hits a blockade, the protein jumps back to its starting position, followed by repetitive cycles of translocations and snapbacks. "This is something that we would never have been able to measure in bulk solution measurement and something that was never seen by any other helicase before," she says.

And the Nobel goes to...

For the Nobel, Myong says she'd like to win for further elucidating helicase function, particularly novel translocation activity.

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