Part:BBa_K3380150
iSpinach fluorescent RNA aptamer
iSpinach is a fluorescent RNA aptamer comprising a short ribonucleotide sequence that exhibits fluorescence upon binding to a specific fluorophore.
Characterization
The iSpinach fluorescent RNA aptamer is derived from the Spinach2 sequence described by [http://www.nature.com/nmeth/journal/v10/n12/full/nmeth.2701.html Strack et al. (2013)]. Figure 1 shows the iSpinach secondary structure folding prediction as simulated by [http://www.nupack.org// NUPACK].
| iSpinach Characteristics | ||
|---|---|---|
| Fluorophore | DFHBI | |
| Excitation wavelength | 442 (nm) | |
| Emission wavelength | 503 (nm) | |
| Quantum Yield | 0.98 | |
| Extinction coefficient | 26100 (M^-1 cm^-1) | |
| Source | [1] | |
Figure 1: iSpinach 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 iSpinach can be used for visualisation by gene fusion to the target of interest. Its fluorescent colour is green. Some of the most commonly used fluorophores are DFHBI ((Z)-4-(3,5-Difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one) and DFHBI-1T ((Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-1-(2,2,2-trifluoroethyl)-1H-imidazol-5(4H)-one). The emission spectra depends on the bound fluorophore.
Its short length allows it to be synthesized rather than being cloned using plasmids. To increase its stability and increase its folding the iSpinach can be transcribed using tRNA scaffolds such as F30 (BBa_K3380101 and BBa_K3380102). The tRNA scaffolds also protect the fluorescent RNA aptamer from degradation by RNAses, therefore increasing its fluorescence.
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]
| None |