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Ústav organické chemie a biochemie AV ČR
The Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences (IOCB Prague) is a leading scientific institution in the Czech Republic, recognized internationally. Its primary mission is basic research in the fields of chemical biology and medicinal chemistry, organic and material oriented chemistry, chemistry of natural compounds, biochemistry and molecular biology, physical chemistry, theoretical chemistry, and analytical chemistry.
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Novel fluorescent probe for real-time imaging of nascent DNA in live cells

Tu, 30.4.2024
| Original article from: IOCB Prague
Novel 2′-deoxycytidine triphosphate (dCTP) nucleotide modified with a thiazole orange fluorophore can be incorporated into DNA strand by DNA polymerases in vitro as well as in live cells.
- **Photo:** IOCB Prague: Novel fluorescent probe for real-time imaging of nascent DNA in live cells
  • Photo: IOCB Prague: Novel fluorescent probe for real-time imaging of nascent DNA in live cells

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.

IOCB Prague: Real-time imaging of in cellulo incorporation of dCOTP into genomic DNA.

This novel approach, published in Angewandte Chemie International Edition journal, can be exploited in studies of genomic DNA organization, dynamics, and function.

Resources

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

Ústav organické chemie a biochemie AV ČR
 

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