Emissive Guanosine Analog Applicable for Real-Time Live Cell Imaging

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.