Talk:CH391L/S12/SpinachRNA

• 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 calculateing the "center" of the fluorescence emission and determine single molecules like when using GFP?
• 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.
• Jeffrey E. Barrick 11:04, 3 March 2012 (EST):Fluorescein-binding aptamers were selected a while ago [1]. There are also rhodamine aptamers that have been used for in vivo imaging apparently [2]. 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?
• 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?

References

1. Holeman LA, Robinson SL, Szostak JW, and Wilson C. Isolation and characterization of fluorophore-binding RNA aptamers. Fold Des. 1998;3(6):423-31. DOI:10.1016/S1359-0278(98)00059-5 |
2. Eydeler K, Magbanua E, Werner A, Ziegelmüller P, and Hahn U. Fluorophore binding aptamers as a tool for RNA visualization. Biophys J. 2009 May 6;96(9):3703-7. DOI:10.1016/j.bpj.2009.01.041 |