Part:BBa_K1368011

Spinach Aptamer 13-2 RNA with Stabilizing tRNA Scaffold

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 exhibit fluorescence under the excitation of ultraviolet light. It’s a good substitute for fluorescent protein in the research in transcription level. The endogenous tRNA-lys can be recognized by the cell and avoid being degraded by ribonuclease. We replace the anticodon region of tRNA-lys into Spinach Aptamer coding sequence to make tRNA Scaffold serve as a camouflage, so the Spinach Aptamer is stabilized.

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]

Usage and Biology

DMHBI was synthesized as outlined inScheme.

N-Acetylglycine (5.26 g, 0.045mol), anhydrous sodium acetate (3.69 g,0.045mol), 4-hydroxy-2,3-dimethoxybenzaldehyde (8.19 g, 0.045mol), and aceticanhydride (15 ml) were stirred at 90℃for 2 h. After allowing the reaction to coolto room temperature, cold ethanol (20 ml) was added while still stirring andthe reaction was left stirring overnight at 4℃.The resultingcrystalline solid was then washed with coldethanol, hot water, hexanesand driedto afford9.65 g (yield 70%) of 3as a pale yellow solid:H NMR (500 MHz,) δ7.56 (s, 2H), 7.18 (s,1H), 3.98 (s, 6H), 2.54(s, 3H), 2.39(s, 3H); LC/MS (LC: gradient 20-95% MeCN[0.1%] over 2.5min, 0.5 ml/minflow rate, MS:): retention time, 2.15 min;purity, 95%; 306.36. Then wecan get Compound 3.Compound 3 (1.12 g, 0.005mol) was refluxedwith 15 ml of ethanol, 1 ml of 40% aqueous methylamine, and 700 mg of potassiumcarbonate for 4h. The reaction mixturewas removed from heat and upon cooling formed an orange precipitate. Theprecipitate containing the product was filtered and washed briefly with coldethanol. The precipitate was then redissolved in a 1:1 mixture of ethyl acetateand 500 mM sodium acetate pH 3.0. The organic layer was separated, dried withanhydrous sodium sulfate and solvent was removed under reduced pressure toyield 717 mg (yield 52%) of DMHBI as an orange solid: 1H NMR (500MHz, DMSO-) δ7.62 (s,2H), 6.90 (s, 1H), 3.80 (s, 6H), 3.09 (s, 3H), 2.34 (s, 3H).DMHBI is easily oxidized. So storagerequires low temperature, dark and inert gas environment.

DH5α cells (TransGen Biotech) were transformed with 10 μl of plasmid DNA expressing chimeras of the human tRNALys3 scaffold fused to the Spinach aptamer sequence. Cells were plated, grown overnight and single colonies were picked for inoculation overnight in Luria Broth containing chloramphenicol. At OD600 = 0.8, 150 μl culture was removed, pelleted and resuspended in 100 μl M9 minimal media and cultivate for 1 h at 37 ℃. Cells were washed twice and incubated with 200 μM DFHBI in M9 media for 5 min. We tested the spinach RNA aptamer in the Tanon 1600R Gel Imaging System under the 302nm, the control groups are the DMHBI and DMHBI + control RNA. The result is below (Fig.1) and we also observed the spinach RNA aptamer by the fluorescence microscope, the result is in the figure 2.

Fig. 1 photographed by Gel Imaging System under the 302nm

Fig. 2 photographed by the fluorescence microscope (40X)

References

Paige J S, Wu K Y, Jaffrey S R. RNA mimics of green fluorescent protein[J]. Science, 2011, 333(6042): 642-646.