Emissive Guanosine Analog Applicable for Real-Time Live Cell Imaging

• Published in: ACS chemical biology Vol. 19; no. 8; pp. 1836 - 1841
• Main Authors: Steinbuch, Kfir B., Cong, Deyuan, Rodriguez, Anthony J., Tor, Yitzhak
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 16-08-2024
• Subjects: Cell Survival - drug effects; DNA-Directed RNA Polymerases - metabolism; Guanosine - analogs & derivatives; Guanosine - chemistry; HEK293 Cells; Humans; Microscopy, Confocal - methods; Viral Proteins
• ISSN: 1554-8929; 1554-8937; 1554-8937
• DOI: 10.1021/acschembio.4c00398

A new emissive guanosine analog CF3thG, constructed by a single trifluoromethylation step from the previously reported thG, displays red-shifted absorption and emission spectra compared to its precursor. The impact of solvent type and polarity on the photophysical properties of CF3thG 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 CF3thG, 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 concentration-gradient conditions, which was suppressed by the addition of guanosine.