Reporter

Part:BBa_K4351010

Designed by: Laura Keffer-Wilkes   Group: iGEM22_Lethbridge_HS   (2022-10-07)


RNA Mango II

RNA Mango forms a G-quadruplex structure and when bound to thiazole orange increases the fluorescence of the dye by several thousand times. With a nanomolar affinity for TOI it can bind to and form a stable complex useful for tracking RNAs during live cell imaging (Dolgosheina et al., 2014).

RNA Mango has been used as a fluorescent tag for tracking small non-coding RNAs (Autour et al., 2018) and live cell imaging (Dolgosheina et al., 2014). Here, we used RNA Mango to look at transcription occurring in real time. The RNA Mango template DNA was mixed with the Promega T7 RiboMax Express kit and incubated with thiazole orange (TOI) in a cuvette. The fluorescence intensity was recorded using a Quanta Master 60 fluorescence spectrometer (Photon Technology International). TOI was excited at 510 nm and the emission spectra recorded between 520 and 600 nm. Readings were taken as soon as the DNA template was added to the reaction, then at 5, 10, 20, 30, 40, 50 and 60 minutes (Figure 3A). As time progressed, the fluorescence intensity increased and then reached a maximum and leveled off. Peak fluorescence at 535 nm was then plotted against time (Figure 3B). The data was then fitted with a logistic growth curve (y = a / (1 + b e-kx ), k > 0) and was found to have a doubling time of 0.1355 min (~8 sec).

resultsfig3.jpg

This new part can be added to other RNA riboswitches or aptamers to potentially make a reporter biosensor for ligand binding. We used this in conjunction with aptamers designed to bind glucose and theophylline and a scramble sequence to act as a negative control.

All three biosensors were unfolded by heating to 95°C. Ligand was then added to the reaction mixture to a final concentration of 20 µM and the RNA was allowed to cool to room temperature. TOI dye was added to the reaction mixture just prior to fluorescence measurements (figure 3).

Interestingly, the glucose biosensor fluorescence increases when glucose is present, almost a two-fold increase compared with the no glucose mixture (figure 3A). The theophylline biosensor however only appears to have a slight increase in fluorescence when ligand was included during the refolding step (figure 3B). The glucose scramble control biosensor, again shows a fluorescent increase in the presence of ligand (figure 3C).

slide9.png

Autour, A., C. Y. Jeng, S., D. Cawte, A. et al. Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells. Nat Commun 9, 656 (2018). https://doi.org/10.1038/s41467-018-02993-8

Dolgosheina EV, Jeng SCY, Panchapakesan SSS, Cojocaru R, Chen PSK, Wilson PD, Hawkins N, Wiggins PA, andUnrau PJ. ACS Chemical Biology 2014 9 (10), 2412-2420 DOI: 10.1021/cb500499x

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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 40


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