The investigation of the organization, function, and dynamics of genomic DNA in chromatin is a challenging topic that can be addressed by fluorescence microscopy. Conventional DNA labeling methods employ metabolically active non-natural nucleoside analogs with subsequent secondary labeling with antibodies or fluorescent tags. But these methods do not allow for truly real-time monitoring of the nascent DNA in live cells due to the background signal of non-incorporated labeled nucleosides abundantly present in the intracellular space.
The researchers led by Tomáš Kraus from IOCB Prague and Michal Hocek from IOCB Prague and Charles University designed and synthesized a novel 2′-deoxycytidine triphosphate (dCTP) nucleotide modified with a thiazole orange fluorophore, which can be incorporated into DNA strand by DNA polymerases in vitro as well as in live cells. Upon incorporation of the modified nucleotide into DNA, the fluorescence of thiazole orange moiety exhibits a shorter lifetime that differs significantly from the intercalated non-incorporated form.
Due to this measurable difference, DNA synthesis in live cells can be monitored in real-time using fluorescence lifetime imaging microscopy. This has allowed the scientists to distinguish and monitor cells which are actively synthesizing DNA from the very first moments after the probe treatment.
This novel approach, published in Angewandte Chemie International Edition journal, can be exploited in studies of genomic DNA organization, dynamics, and function.
Read the paper: Kuba, M.; Khoroshyy, P.; Lepšík, M.; Kužmová, E.; Kodr, D.; Kraus, T.; Hocek, M. Real-time Imaging of Nascent DNA in Live Cells by Monitoring the Fluorescence Lifetime of DNA-Incorporated Thiazole Orange-Modified Nucleotides. Angew. Chem. Int. Ed. 2023, e202307548. https://doi.org/10.1002/anie.202307548
