Multiphoton characterization and live cell imaging using fluorescent adenine analogue 2CNqA

Nilsson, Jesper; Benítez-Martín, Carlos; Sansom, Henry  G; Pfeiffer, Pauline; Baladi, Tom; Le, Hoang-Ngoan; Dahlén, Anders; Magennis, Steven W.; Wilhelmsson, L. Marcus

doi:10.1039/d3cp01147j

Cited by 4 publications

(2 citation statements)

References 30 publications

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“…T7 RNA polymerase was found to initiate in vitro transcription with CF3th G, resulting in highly emissive 5′-end-tagged RNA transcripts, suggesting that the new emissive analog, despite its large Hoogsteen face substituent, can still be recognized by polymerases. While other emissive nucleosides have been recently reported to facilitate live cell imaging, it appears that these analogs could be visualized only when embedded into RNA strands. − Here, we demonstrate that due to its intense visible emission, CF3th G can be visualized as the free nucleoside in live mammalian cells and could be monitored in real time. Imaging experiments with and without guanosine allude to its similarity to guanosine.…”

Section: Resultsmentioning

confidence: 99%

Emissive Guanosine Analog Applicable for Real-Time Live Cell Imaging

Steinbuch,

Cong,

Rodriguez

et al. 2024

ACS Chem. Biol.

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A new emissive guanosine analog CF3th G, constructed by a single trifluoromethylation step from the previously reported th G, displays red-shifted absorption and emission spectra compared to its precursor. The impact of solvent type and polarity on the photophysical properties of CF3th G suggests that the electronic effects of the trifluoromethyl group dominate its behavior and demonstrates its susceptibility to microenvironmental polarity changes. In vitro transcription initiations using T7 RNA polymerase, initiated with CF3th G, result in highly emissive 5′labeled RNA transcripts, demonstrating the tolerance of the enzyme toward the analog. Viability assays with HEK293T cells displayed no detrimental effects at tested concentrations, indicating the safety of the analog for cellular applications. Live cell imaging of the free emissive guanosine analog using confocal microscopy was facilitated by its red-shifted absorption and emission and adequate brightness. Real-time live cell imaging demonstrated the release of the guanosine analog from HEK293T cells at concentrationgradient conditions, which was suppressed by the addition of guanosine.

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Section: Resultsmentioning

confidence: 99%