Flipping off HIV Reverse Transcriptase

Get out your crap hat!* Former Blockhead Elio Abbondanzieri (and a veritable horde of Zhuang lab grad students) has published a paper in Nature on HIV reverse transcriptase. Reverse transcriptase (RT) has two opposite activities: polynucleotide synthesis, and degradation. The active sites are at opposite ends of the molecule, so how it knows when to do what is a big open question. By labeling the RT and its substrates, they were able to detect the binding orientation of the RT, and thereby show that it binds in different orientations to elongation substrates and degradation substrates. Further, the use of a single molecule assay allowed them to assay rate constants, and show that occasionally RT will "flip" from one orientation to the other, and that these flipping rates can be affected by small molecules, such as Nevirapine, a drug which targets the HIV RT.

The paper made me wonder about optical trapping of RT to look at its processivity, but this has apparently been tried (by Elio, in fact), and its processivity is too low to make it worthwhile. I still wonder whether some kind of "fishing" assay could be developed, where multiple brief binding events could be recorded by dangling a bead with RT near a surface with substrate, or something like that.

*Local tradition has it that Elio was famous for declaring, during journal clubs, that he was getting out his "crap hat" in response to what he perceived as sub-par science.

I think I made a mistake...

Why did I spend six and a half years taking classes, passing quals, taking data, and publishing papers, just to get a doctoral degree in physics, when I could have gotten a doctoral degree in Parapsychic Science from the comfort of my own home! And probably in way less time too. Oh, wait, now I remember: I did it for the babes.

Huzzah!

My F32 fellowship was officially funded! So I brought beer and chocolate to the lab to celebrate. The Block lab tradition is to drink shots to celebrate such things, but Steve is off getting inducted into the National Academy, so I took the opportunity to have beer instead, because, really, who the hell wants to drink hard liquor at 4:30 in the afternoon? I brought Anchor Summer Beer and He-Brew Genesis Ale*.

*I have a theory that physicists love to talk about beer and drink beer in order to compensate for the fact that they are not engaged in the most rugged or masculine profession. It's technically masculine, in the sense that it is dominated by men. But, it lacks the musk and sweat of, say, being a lumberjack, or a stock car racer. Or even a salesman, for that matter.

Nature Photonics

I used to consider myself an optics jock, but these days, my friends call me "gel monkey" instead, which says a lot about the way my work is going. But, reading Nature Photonics has really made me hanker for my optics jock days. I had no idea this was such a cool journal! A couple of neat articles:

• We had an inquiry about a visit from the Degiorgio lab at the University of Pavia in Italy, and she pointed me to an article of theirs about a new fiber-optic optical tweezer. (Unfortunately, it doesn't look like this is getting indexed by Pubmed, which is a shame, since that's where I go for most of my searching these days.) By using an annular fiber, or a fiber tweezer, they reduce the amount of axial force on the particle (by getting rid of those useless low-NA rays from the middle) and can therefore achieve a much higher effective NA at longer working distances.

• This is a really cool article about using nanofabricated aluminum optical antennas for actively directing radiation from single fluorophores. I seriously had this idea like 10 years ago, after hearing Joe Lackowicz speak about metal-enhanced fluorescence at BPS. At the time, we were trying to directly detect rotation of the S4 segment of voltage-gated potassium ion channels during gating, to follow up on this paper. We were trying to use polarized FRAP, but couldn't get it to work after several years of trying. I thought that another way to detect rotation would be to probe a single molecule with a metal tip, and detect changes in intensity as the dipole rotated with respect to the tip. Anyway, I was ahead of my time, but this looks really neat. In some sense, this is an extension of the optical orientation imaging techniques developed by Robert Dickson and Jörg Enderlein. It's a nice new approach though, and I think it could be highly interesting as it develops.

On an unrelated note, it looks like the Selvin lab (my graduate thesis lab) has finally gotten their optical trap up and running. Hooray! It's been many years in the works.

New Reviews

A couple of nice new reviews that have just come out in Annual Review of Biochemistry:

• T.J. Ha has published a review of single molecule techniques titled Advances in Single-Molecule Fluorescence Methods for Molecular Biology. It covers a lot of ground, and looks like a great primer for anybody new to the field.
  
• Our lab also has a review out, Single-Molecule Studies of RNA Polymerase: Motoring Along, synthesizing some of the more recent results on RNA polymerase studies using optical trapping and other approaches.

Science in fraudulo

Those wacky Koreans have been at it again! Apparently two papers about an almost unbelievably ironically named technique called "MAGIC" have been found to "lack scientific truth". The technique purportedly allowed the identification of drug targets by binding drugs to magnetic nanoparticles which were then flowed into cells. They would then bind to target proteins, and the beads could be recovered magnetically. Supposedly. It turns out the images were faked, and there was never any data. Luckily, the Korean Ministry of Science and Technology had procedures laid out for investigating such cases, put in place after the last time this happened.

What's really amazing, though, is that this clown went and actually tried to commercialize his non-existent technology by founding a company called CGK. How did he suppose this was going to work, inviting somebody to reproduce your irreproducible results for money? In any case, we all know what happens when you rely on magic to run your technology.

My only question, though, is: does this qualify as single-molecule news just because it uses magnetic nano-particles? Or am I totally out of my milieu with this one?

Video rate stimulated emission depletion (STED)

Stefan Hell has published a new paper on video-rate tracking of single synaptic vesicles in axons using STED. STED is an imaging method that gives an improvement over the usual diffraction limit of λ/2NA by a factor of 3 - 4*. STED achieves this improvement by using a pulsed probe beam, followed by a pulsed "depletion" beam. By using diffraction, the depletion beam can be contoured like a donut around the imaging beam, and can induce stimulated emission in a region surrounding it. The remaining excited fluorophores now have a reduced spatial extent, and by collecting fluorescence that occurs after the depletion beam, the effective point spread function size is reduced.

This paper is the first example of using STED for video tracking, as opposed to imaging of static objects, and it's a nice application. But STED hasn't been very widely adopted in general, for two basic reasons, in my opinion:

1) It is incredibly complicated to implement. It requires two co-aligned lasers, which have to be spatially and temporally modulated in sync. I saw Stefan Hell speak about this work at UIUC (I believe he was there giving a job talk), and I remember thinking that there was no way on God's green earth that anybody else was going put in the effort to duplicate this. Now, this is coming from someone who works in an optical trapping lab, where "aligning obscenely complicated optics" is part of breakfast, and this hasn't stopped plenty of people from building optical traps. But, see below.

2) A factor of four improvement just isn't all that great. From a single-molecule perspective, this is similar to one of the problems that I saw with SHRImP: Most macro-molecular complexes are about 10 nm in size max, which is within the range of FRET and below the range attainable by these types of "diffraction-limit-improved" approaches. On the other hand, most cells are at least a micron, and eukaryotic cells, which have internal structures, are typically more like 10 - 50 microns. Most of these structures can be imaged perfectly well with conventional microscopy, and there's not a huge number of interesting things too big to measure with FRET and too small to measure with conventional confocal scanning. Now, I obviously have a physicist's bias here, and I'm sure there are plenty of people who know more cell biology than I who would disagree**. But, the fact is that I don't really see people rushing to implement STED in their laboratories.

Now, optical trapping requires probably as much or more complication than STED, at least the sorts of super fancy optical traps we have here in the Block lab. But the difference, in my mind, is that optical trapping gives you an entirely new tool for measuring and manipulating. It's completely orthogonal to fluorescence approaches, and therefore it has an immense amount of power to go where other techniques cannot even hope to tread. STED has all the complication, but far fewer benefits as a result. Furthermore, optical trapping naturally appeals to physicists, with its emphasis on forces and mechanical transitions. STED is a tool that would primarily appeal to cell biologists, but it requires a physicist to build it. Hence, there is probably a dearth of people who both a) have questions that STED can answer, and b) have the expertise to actually build such an instrument.

On the other hand, it looks like you can now buy a commercial off-the-shelf STED microscope from Leica, so who knows? Maybe this will be the next big thing.

*They claim the diffraction limit is about 260 nm. With a typical oil immersion objective, NA = 1.45, and Cy3/TAMRA is probably one of the most popular dyes, with an emission peak of around 570 nm, such that the diffraction limit = 570/(2*1.45) = 197 nm. However, in this paper, they used Atto 647 (emission peak = 673 nm) and a 1.4 NA objective, which would result in a diffraction limit of 673/(2*1.4) = 240 nm. I believe they're calculating the "diffraction limit" in this case as the FWHM of the observed point spread function (PSF). But, there are two sort of tricky things here to note. The first is that their claim of a 260 nm diffraction limit, instead of the more typical 200 nm, makes their data look better by contrast. The second is that they claim an 18-fold improvement over the diffraction limit, but this is in terms of focal area, not point spread function width. This is also a tricky way to make their numbers seem a bit better than they are, because the interesting parameter is the linear length scale, which tells you how far apart two objects can be and still be resolved. They only reduce the point spread function width itself by a factor of about 4.

**Although I did just organize a Fat Alberts club here in the Block lab so us ignorant physicists can start learning more about cell biology. I just presented chapter three, "Proteins", last week, and we're on to DNA this week. Woo!