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FRET and the Single Molecule

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  • Title: HHMI Investigator and Associate Professor, Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign
  • Education: PhD, University of California, Berkeley, 1996; Postdoc, Stanford University and the Lawrence Berkeley National Laboratory
  • Recommended by: Gene Robinson

Taekjip Ha's work sits at the fore of experimental bio-physics, and that's been the case ever since he broke new ground on single-molecule fluorescence resonance energy transfer as a graduate student. Trained as a physicist, Ha didn't exactly aim to get the 'bio' prefix added to his PubMed results. “Back then I wasn't interested in biology at all,” Ha recalls. “I was just using DNA as a way of putting the two dyes close to each other so I could measure this transfer.”

Measure it he did in Scott Weiss' lab at the University of California, Berkeley; by the last year of his PhD, Ha's work resulted in a paper on single-molecule FRET in PNAS. Despite getting into print, Ha was skeptical of FRET's biological applications. In fact, he “didn't believe a word of it” when Weiss suggested in the paper's abstract that the technique could be used to measure the conformational dynamics of single molecules. “I was wrong,” laughs Ha, whose current work builds on just that.

Ha's research draws on physical concepts to tease out the answers to decidedly biological questions. In terms of helicase, for instance, Ha is working to understand directionality of the enzyme by physical and computational approaches. The endgame is the “rational design of a mutant that will go backward” on a single strand of DNA as a means to understanding why helicases move in one direction versus another.

Ha is also interested in extending his work by “combining single-molecule FRET imaging with optical tweezers,” by which small forces can be applied very precisely. The dynamics of DNA molecules can be altered dramatically when force is applied, says Ha, whose group is studying such fluctuations with a model system known as a Holliday junction, an intermediate made up of four strands of DNA. “What's unique about our approach is that we're actually measuring the effects of force by fluorescence,” he says.

Looking ahead

Right now, Ha's lab is working on “extreme in vitro” studies, but Ha would like to see the field move toward doing all measurements in a single cell. Difficulty in measuring conformational changes at the single-cell level has been in the probes. Fluorescent dyes on the market are not bright enough, and they don't have staying power. “There's tremendous background fluorescence and they photobleach very quickly,” Ha notes. So although one can perform imaging in individual cells with conventional fluorescence, it's not ideal, as fluorophores themselves limit the information you can glean from single-molecule measurements.

Quantum dots are another possibility — they're bright enough and Ha's team has already succeeded in making them non-blinking — but the problem is they're just too big for the sort of techniques Ha has in mind. Once layered with materials like polymer to make the dots water-soluble and streptavidin to make them attach to proteins, a fully loaded quantum dot could be about 200 Å. “If someone makes a next-generation quantum dot that is small, water-soluble, and bioconjugable, that would be a dream,” Ha says.

Publications of note

Two recent publications by Ha's team highlight topics that are “really new and of biological interest,” he says. These include results concerning a bacterial helicase and its sometimes snappy travels along single-stranded DNA, which the team reported in Nature last year. More recently, in a Cell paper entitled “Real-time observation of RecA filament dynamics with single monomer resolution,” Ha and colleagues used single-molecule fluorescence assays and hidden Markov modeling to investigate the growth and goings-on of RecA and its homologues.

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