- Peter Otoupal 22:33, 1 March 2012 (EST):Is it possible to use this chromophore-aptamer complex fluorescence technique to distinguish localized sites of RNA activity in cells? This picture makes it seem like the entire E. coli cell is caused to fluoresce when Spinach is expressed. It seems like a great technique; I guess it just seems like Spinach's strength would be in pinpointing RNA activity within cells, and it's hard to tell if such precision is possible from that picture.
- David M. Truong 22:50, 1 March 2012 (EST):Going with what Peter said, what is its potential for single-molecule use? Is the signal too diffuse (and the background too high)? Does it have potential for calculating the "center" of the fluorescence emission and determine single molecules like when using GFP?
- Jon M. Laurent 14:45, 5 March 2012 (EST):Yes, it is definitely possible to localize RNA within cells. I didn't discuss it much, but I did show one figure from the supplemental material of the original paper that localizes the 5S rRNA in human cells to small granules under certain condition. There is also claims from the same lab that they are already doing localization work with SpA in neuronal cells, although that is as of yet unpublished. The appearance of the whole cell being lit up is due mostly to the fact that transcription is being driven by a very strong promoter, which basically floods the cell with lots of the SpA transcript. Additionally, said transcript has no localization signals, so it will just float freely around the cytoplasm, giving it the appearance of lighting up the whole cell. For David, there is definite potential for doing single molecule stuff, I think, although I can't claim to know much about the details of single-molecule microscopy. The original lab does have claims that they are using SpA and derivatives to do Protein/RNA FRET studies in neurons. The similarities to GFPs spectral properties help in this regard, and should potentially make it useful for use anywhere GFP has been used similarly. (I'm also currently working on methods for visualization RNA localization with SpA in lab)
- Yi Kou 09:09, 3 March 2012 (EST):what about the turnover of the Spinach? Along with David's question, I think for single molecule application, such as sm-FRET, Spinach is still far from satisfying, possibly from its inability to maintain good intentional photobleaching and low yield of brightness. You would probably get fluctuating weak signals that are hard to tell the difference from background 'blinking', as compared to other labeling methods. But it is of great potential in the future for sure.
- Jon M. Laurent 14:45, 5 March 2012 (EST):While I don't know much about the details of single-molecule microscopy, you do make a valid point. I think the lack of photobleaching is both useful and detrimental to the utility of SpA in these regards, as it enables visualization for long periods of time, but does not allow for certain types of studies to be undertaken that require bleaching (fluorescence recovery, measuring new RNA production, etc.). It's speculated that the lack of photobleaching is actually an effect of the rapid dissociation/re-association of the SpA RNA with the DFHBI ligand, so that could be taken advantage of for difference methods of bleaching, such as simply washing out the molecule and re-doping. As mentioned above, the lab that developed SpA claims to be doing protein/RNA FRET right now with SpA, so it is apparently possible. Turnover of SpA itself is unknown, and would presumably be tied to whatever RNA is tagged with it, or in the case of expression by itself, whatever turnover is typical of T7 transcripts.
- Jeffrey E. Barrick 11:04, 3 March 2012 (EST):Fluorescein-binding aptamers were selected a while ago . There are also rhodamine aptamers that have been used for in vivo imaging apparently . The main advantage of Spinach is that the background is so low for the free fluorophores floating around in a cell that are not bound to the RNA, right?
- Jon M. Laurent 14:45, 5 March 2012 (EST):Some of these are mentioned in the SpA paper, yes. The background problems with of some of these other molecules have various origins, most of which are not inherent in the GFP-like fluorophores. Along with what you mention about background, some of the other chromophores have shown cytotoxicity, and of course the fact that GFP fluorescence is well-understood and easily visualized with existing tools that are already present in most labs. I didn't read those papers in much detail, so I can't say anything about the brightness of those particular labels, either. Although the brightness of SpA is not ideal, it may be 'better' than those mentioned, as it is brighter than many fluorescent proteins besides GFP, I believe.
- Yi Kou 11:17, 3 March 2012 (EST):yes, I think it is great for this aspect of in vivo imaging
- Joe Hanson 16:39, 3 March 2012 (EST): In class I mentioned this in vitro labeling technique that's marketed by Mirus Bio: Label IT. It's a non-specific DNA or RNA label that can be conjugate to many dyes or secondary binders. I know from personal experience that it inhibits some biological activity, but it is silly simple and works well. Any thoughts on it?
- Jon M. Laurent 14:45, 5 March 2012 (EST):I'd never seen or heard of this before, thanks for the heads-up. This seems like a good method for visualizing certain RNAs, most notably small ones, such as siRNAs and microRNAs, or plasmids, as they mention. I don't see it having much overlap in utility with something like SpA, as it is nonspecific, the label is attached in vitro, and the target is modified covalently with the fluorophore. SpA is obviously not a good choice for microRNAs or similar because of it's size and structure, but it seems like the better choice to label pretty much any of larger RNA in vivo.
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