Difference between revisions of "Part:BBa K3380153"

Revision as of 04:04, 6 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].

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

Contribution

• Group: SCU-China iGEM team 2020
• Author: Yilong Xu
• Summary: compare the strength of this promoter and pGAP.