Difference between revisions of "Part:BBa K3380150"
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iSpinach is a fluorescent RNA aptamer comprising a short ribonucleotide sequence that exhibits fluorescence upon binding to a specific fluorophore.  
 
iSpinach is a fluorescent RNA aptamer comprising a short ribonucleotide sequence that exhibits fluorescence upon binding to a specific fluorophore.  
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===Characterization===
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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].
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{| style="color:black" cellpadding="6" cellspacing="2" border="2" align="right"
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! colspan="3" style="background:#C0C0C0"|'''iSpinach Characteristics'''
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|-
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|'''Fluorophore'''
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|DFHBI
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|-
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|'''Excitation wavelength'''
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|442 (nm)
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|-
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|'''Emission wavelength '''
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|503 (nm)
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|-
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|'''Quantum Yield'''
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|0.98
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|-
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|'''Extinction coefficient'''
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|26100 (M^-1 cm^-1)
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|-
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|'''Source'''
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|[https://academic.oup.com/nar/article/44/6/2491/2499459]
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|}
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[[File:Part_BBa_K3380150.png|400px|iSpinach folding]]
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'''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===
 
===Usage and Biology===
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It is usually used in live-cell imaging, however it can also be used in vitro. 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. The [https://doi.org/10.1093/nar/gkw083/ DFHBI]fluorophore has an excitation wavelength of 492 (nm) and an emission wavelength of 516 (nm).
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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.  
  
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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 increases its fluorescence.  
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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 ([https://parts.igem.org/Part:BBa_K3380101 BBa_K3380101]  and [https://parts.igem.org/Part:BBa_K3380102 BBa_K3380102]). The tRNA scaffolds also protect the fluorescent RNA aptamer from degradation by RNAses, therefore increasing its fluorescence.  
  
 
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Latest revision as of 16:26, 23 October 2020


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]

iSpinach folding

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]