Part:BBa_K1368010
Spinach Aptamer 13-2 RNA driven by c1 promoter
Spinach Aptamer 13-2 RNA is a small RNA with specific stem loop structure which can combine with DMHBI, a kind of fluorescein, and then the RNA-fluorophore complexes will fluoresce under the excitation of ultraviolet light. We use cI promoter to derive Spinach Aptamer 13-2 RNA that the part will serve as a reporter in transcription level under control of cI protein which acts as an input signal.
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]
Usage and Biology
Imaging RNA in living cells is important for understanding the function and regulation of divece classes of cellular RNAS. There are some aptamers like Spinach and dBroccoli,witch bind and activates the fluorescence of fluorophores similar to that found in green fluorescent protein. In our project, At first, we use GFP as a reporter of our oscillator, but there is a long delay from DNA to protein. We think it’s better to shorten the delay. Luckily, there is a RNA aptamer named dBroccoli that can be used for the genetic encoding of fluorescent RNA. The dBroccoli binds and activates the fluorescence of (Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-1-(2,2,2-trifluoroethyl)-1Himidazol-5(4H)-one) (DFHBI-1T).
DMHBI was synthesized as outlined inScheme.
800px （荧光素反应1） （荧光素反应2）
N-Acetylglycine (2.22 g, 18.99 mmol), anhydrous sodium acetate (1.56 g, 18.99 mmol), 4-hydroxy-3,5-difluorobenzaldehyde (3.00 g, 18.99 mmol), and acetic anhydride (7.2 ml) were stirred at 100 ºC for 1 h. After allowing the reaction to cool to room temperature, cold ethanol (30ml) was added while stirring and the reaction was left stirring overnight at 4 ºC. The resulting crystalline solid was then washed with a small amount of cold ethanol, hot water, hexanes and dried to afford 3.40 g (yield 64%) of 1 as a pale yellow solid。Compound 1 (472 mg, 1.68 mmol) was refluxed with 4 ml of ethanol, 250 mg (2.52 mmol) of 2,2,2-Trifluoroethylamine, and 348 mg of potassium carbonate for 4 h. The reaction mixture was removed from heat and cooled to room temperature. The solvent was evaporated and the mixture was redissolved in a 1:1 mixture of ethyl acetate and 500 mM sodium acetate pH 3.0. The organic layer was separated, dried with anhydrous sodium sulfate. The solvent was removed under reduced pressure and the reaction mixture was purified by column chromatography (CH2Cl2 : MeOH = 10:1) to afford 215 mg (yield 40%) of 12 as yellow solid。
DH5α cells (TransGen Biotech) were transformed with 10 μl of plasmid DNA expressing dBrpccoli. Cells were plated, grown overnight and single colonies were picked for inoculation overnight in Luria Broth containing chloramphenicol. At OD600 = 0.8, 200 μl culture was removed, pelleted and resuspended in 200 μl M9 minimal media with 200 μM DFHBI-1T and cultivate for 40 min at 37 ℃. we observed the spinach RNA aptamer by the fluorescence microscope, the result is in the figure 1.
图片
References
Wenjiao S, Strack R L, Nina S, et al. Plug-and-play fluorophores extend the spectral properties of Spinach.[J]. Journal of the American Chemical Society, 2014, 136(4):1198-1201. Filonov G S, Moon J D, Nina S, et al. Broccoli: Rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution.[J]. Journal of the American Chemical Society, 2014, 136.
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