Tetramolecular fluorescence complementation
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Lizenz | CC-Namensnennung - keine Bearbeitung 4.0 International: Sie dürfen das Werk in unveränderter Form zu jedem legalen Zweck nutzen, vervielfältigen, verbreiten und öffentlich zugänglich machen, sofern Sie den Namen des Autors/Rechteinhabers in der von ihm festgelegten Weise nennen. | |
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00:05
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06:24
Blätterteig
Transkript: Englisch(automatisch erzeugt)
00:15
Hello, my name is Stefanie Kellermann and this is Anna Rath and we are both PhD students at the Institute of Biochemistry and Molecular Biology.
00:23
In our research group we are interested in novelle labelling strategies for RNA. We want to design a probe that binds sequence specifically, lights up upon binding and can be produced by the cellular machinery. One approach that meets all these criteria is the split GFP system combined with the RNA binding protein Pummelio as realized by the OSARBA group.
00:43
Here the green fluorescent protein from the jellyfish Echorea vectoria is split into two halves that reassemble upon binding of the two Pummelio protein to the same target RNA. However, the traditional split GFP can easily self-assemble and therefore cause a high fluorescent background.
01:03
To overcome this limitation we designed the Tetra molecular fluorescence complementation system. And now we will go to the lab and show you how the system works. This system relies on the binding of two different Pummelio proteins to one target RNA.
01:21
To gain a fluorescent signal upon binding of these proteins we use the three-body split GFP created by the Waldo group. To create the split GFP, two beta strands of the full beta barrel of GFP are detached. The resulting GFP detector is non-fluorescent although correctly folded.
01:40
The two beta strands are now fused to the Pummelio proteins as you can see here. Upon binding of these two Pummelio proteins to the target RNA in close proximity and the correct orientation, the two beta strands together with the GFP detector can assemble and fluorescence is reconstituted.
02:04
In contrast, if one of the binding motifs is missing or modified, only one Pummelio protein can bind and no fluorescence occurs. After we created the Pummelio fusion proteins, large amounts can easily be produced inside E. coli cells and purified using affinity chromatography.
02:27
Using these two proteins, Stephanie now prepares a measurement to detect specific RNAs. She incubates the Pummelio proteins with the target RNA box AB at equimolar concentrations. One reaction containing the negative control RNA box AC is prepared as well.
02:45
During a 30-minute incubation at room temperature, the Pummelio proteins can bind to their target RNA. After complex formation of the Pummelio proteins and the RNA, Stephanie now adds the GFP detector to start complementation.
03:02
Here you can see the difference between the GFP detector which is non-fluorescent and the full protein. Since we want to monitor the whole complementation process, we start measuring fluorescence intensity immediately. We measure fluorescence intensity with this plate reader.
03:29
Excitation occurs at 488 nm and emission is measured at 530 nm. I will now start the measurement which will take several minutes.
03:40
This is how the measurement looks like after 20 minutes. As you can see, the signal for the positive control RNA box AB can already be distinguished from the negative control RNA box AC after 10 minutes. To show you how a completed measurement looks like, we prepared one for you where we measured overnight.
04:01
You can see that normally after 5 to 10 hours, the signal for the box AB RNA reaches a maximum and is substantially higher than the negative control. This figure shows the various controls we measured, including RNAs with switched modules as well as DNA targets. Positively, detection of the RNA was also possible in presence of competitor RNA or cell lysate.
04:27
To visualize the reconstituted protein on a gel, Marina now adds loading dye to the samples and transfers them onto a non-denaturing protein gel.
04:54
The gel now runs at 100 V until the proteins are separated.
05:13
The gel is now prepared for imaging of the fluorescent proteins.
05:21
Using this fluorescence imager, we can make those protein bands visible that contain the reconstituted GFP. To do so, the gel is illuminated using blue LEDs which excite the GFP. Here, the GFP bands are very prominent in the reactions that contain the correct target RNA.
05:42
In the other lanes, with the negative controls, no fluorescence is detectable. As we have shown you today, tetramolecular fluorescence complementation is a powerful means to detect specific RNA. After sequence-specific target binding and subsequent GFP complementation, the signal is up to 70-fold higher than the background.
06:02
The importance of the correct orientation and presence of both familiar binding motifs was clearly demonstrated. Since all components could be produced by the cellular machinery, our system should also work in vivo. We anticipate that our system can be applied to monitor a wide range of target RNAs. Thank you for watching our video and goodbye from Hamburg!