Tracking mRNA within living cells is crucial for understanding RNA biology and improving the development of RNA-based therapeutics. Traditional fluorescent labels such as Cyanine dyes (e.g. Cy3 or Cy5), while useful, often disrupt the natural structure and function of RNA due to their bulky size. Now, a new approach offers a promising alternative: tCO – a fluorescent tricyclic cytosine analogue (Figure 1A & B).
In a breakthrough study, Baladi et al. have demonstrated that tCOTP (the triphosphate derivative of tCO) can be enzymatically incorporated into mRNA by 3’-end labeling with terminal deoxynucleotidyl transferase (TdT) or during in vitro transcription with up to 100 % tCOTP/CTP substitution (Figure 1C)[1]. Unlike conventional dyes, tCOTP maintains normal base pairing and stacking, resulting in minimal disruption to mRNA's natural structure and function. This allows scientists to visualize mRNA in cells without interfering with important biological processes. Extensive testing showed that tCO-labeled mRNA is efficiently translated into correctly folded and localized proteins, both in vitro and in live cells (Figure 1D & E). This native-like labeling technique allows researchers to track mRNA delivery, stability, and protein synthesis dynamics in real time, providing valuable insight into RNA biology.
Figure 1: Efficient incorporation of the fluorescent tCOTP preserves RNA functionality and protein translation (according to Baladi et al. (2021)).
A) Structural comparison of Cytidine 5'-triphosphate (CTP) and fluorescent tricyclic cytosine analogue (tCOTP).
B) Picture of UV-light-irradiated solution of purified TdT3 RNA end-labeled with tCO.
C) Incorporation of tCO into full-length mRNA by T7 RNA polymerase. RNA probes were generated by in vitro transcription using increasing percentages of tCOTP/CTP substitution (0 - 100 %). The products were analyzed by agarose gel electrophoresis, with labeled RNA was visualized directly and total RNA detected using RNA intercalator ethidium bromide (EtBr). L: ladder.
D) Cell-free translation of calmodulin-3 (CALM3) mRNA. Western blot (WB) of the in vitro translation
reactions. NTC: no template control, +: kit template RNA control, L: ladder.
E) Translation and visualization of tCO-labeled mRNA in human SH-SY5Y cells. Snap-shot images from a confocal time-lapse experiment to monitor the intracellular trafficking of 75 % tCO-labeled mRNA (red) introduced, by chemical transfection, into cells with an overexpression of mRFP-Rab5 to label early endosomes (orange). Resulting expression of H2B-GFP protein in the nucleus is shown in green.
| Nucleotide | Cat. No. | Amount | Price |
|---|---|---|---|
| tCOTP | NU-315 | 10 µl (10 mM) | 990,00 € |
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[1] Baladi et al. (2021) Stealth Fluorescence Labeling for Live Microscopy Imaging of mRNA Delivery. J. Am. Chem. Soc. 143 (14):5413.
