Difference between revisions of "Part:BBa K3380153"
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Latest revision as of 21:39, 21 October 2021
Broccoli fluorescent RNA aptamer
Broccoli is a fluorescent RNA aptamer comprising a short ribonucleotide sequence that exhibits fluorescence upon binding to a specific fluorophore.
Characterization
The Broccoli aptamer was designed and described by Filonov et al. (2014). 'Figure 1 shows the Broccoli secondary structure folding prediction as simulated by [http://www.nupack.org// NUPACK].
| Broccoli Characteristics | |
|---|---|
| Fluorophore | DFHBI-1T |
| Excitation wavelength (nm) | 472 |
| Emission wavelength (nm) | 507 |
| Quantum Yield | 0.94 |
| Extinction coefficient (M^-1 cm-1) | 29600 |
| Source | [1] |
Figure 1: Broccoli secondary structure prediction. The adenine (A), cytosine (C), guanine (G) and uracil (U) nucleobases are shown in green, blue, black and red respectively.
Usage and Biology
It is usually used in live-cell imaging, however it can also be used in vitro. The Broccoli aptamer can be used for visualisation by gene fusion to the target of interest. Its fluorescent colour is green. The most commonly used fluorophore in tandem with Broccoli aptamer is DFHBI-1T ((Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-1-(2,2,2-trifluoroethyl)-1H-imidazol-5(4H)-one).
Its short length allows it to be synthesized rather than being cloned using plasmids. To increase its stability and increase its folding the Broccoli can be transcribed using tRNA scaffolds such as F30 (BBa_K3380101 and BBa_K3380102). The tRNA scaffolds protect the fluorescent aptamers from the degradation by RNAses, therefore the scaffolds increases their fluorescence. However, Broccoli can be successfully used without a scaffold.
To increase its fluorescence, the Broccoli dimerization has been successfully attempted, exhibiting almost twice the fluorescence of the monomeric aptamer.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Contribution
- Group: SCU-China iGEM team 2021
- Author: Yilong Xu
- Summary: Improve this part by expanding itself
- 'Link to our biobrick: https://parts.igem.org/Part:BBa_K3977006
Characterization/Improvement
Based on BBa_K3380153, we construct a RNA adaptor with 4 repeats of BBa_K3380153, We put them under J23100, and detect their performance in 488/527.
Our result shows that BBa_K397706 show more output (C) , meaning that it performed better than original part BBa_K3380153 and our improvement is successful.
Figure 1: Relative fluorescence intensity BBa_K3380153 and BBa_K3977006