Flow cytometric determination of cell cycle progression via direct labeling of replicated DNA

Kužmová, Erika; Zawada, Zbigniew; Navratil, M.; Günterová, Jana; Kraus, Tomáš

doi:10.1016/j.ab.2020.114002

Cited by 8 publications

(11 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The analyses of the first fraction revealed that the two expected populations of cells (G1 and G2/M phase non-labeled vs S phase labeled) are indeed differentiated, but the peaks of fluorescence intensities were separated by one order of magnitude, which is lower than what we observed in our recent work. 6 The control experiments revealed that the background fluorescence in non-synthesizing cell populations (G1 and G2/M) was relatively high, thus decreasing the separation of the cell populations. Moreover, measurements of the second fraction of cells revealed that the addition of DRAQ5 caused lowering of fluorescence intensity of the S-phase cells.…”

Section: ■ Synthesismentioning

confidence: 99%

“…A novel one-step labeling protocol was reported that relies on the incorporation of metabolically active fluorescently labeled deoxynucleoside triphosphates (FL-dNTPs) into genomic DNA in live cells. This method − is rapid and operationally simple, and it does not require permeabilization of the cell membrane; polar and negatively charged FL-dNTPs are delivered into the cells by the synthetic nucleoside triphosphate transporter ( SNTT1 ), and the unreacted residual material is exported by the cells themselvespresumably by efflux pumps. This natural clearance mechanism allows us to acquire images of labeled DNA with a high signal-to-noise ratio in live cells within less than 1 h after their treatment, without the need for fixation and washing of the cells.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

trans-Cyclooctene- and Bicyclononyne-Linked Nucleotides for Click Modification of DNA with Fluorogenic Tetrazines and Live Cell Metabolic Labeling and Imaging

et al. 2023

Self Cite

View full text Add to dashboard Cite

A series of 2′-deoxyribonucleoside triphosphates (dNTPs) bearing 2-or 4-linked trans-cyclooctene (TCO) or bicyclononyne (BCN) tethered through a shorter propargylcarbamate or longer triethyleneglycol-based spacer were designed and synthesized. They were found to be good substrates for KOD XL DNA polymerase for primer extension enzymatic synthesis of modified oligonucleotides. We systematically tested and compared the reactivity of TCO-and BCN-modified nucleotides and DNA with several fluorophore-containing tetrazines in inverse electron-demand Diels−Alder (IEDDA) click reactions to show that the longer linker is crucial for efficient labeling. The modified dNTPs were transported into live cells using the synthetic transporter SNTT1, incubated for 1 h, and then treated with tetrazine conjugates. The PEG3-linked 4TCO and BCN nucleotides showed efficient incorporation into genomic DNA and good reactivity in the IEDDA click reaction with tetrazines to allow staining of DNA and imaging of DNA synthesis in live cells within time periods as short as 15 min. The BCN-linked nucleotide in combination with TAMRA-linked (TAMRA = carboxytetramethylrhodamine) tetrazine was also efficiently used for staining of DNA for flow cytometry. This methodology is a new approach for in cellulo metabolic labeling and imaging of DNA synthesis which is shorter, operationally simple, and overcomes several problems of previously used methods.

show abstract

Section: ■ Synthesismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

trans-Cyclooctene- and Bicyclononyne-Linked Nucleotides for Click Modification of DNA with Fluorogenic Tetrazines and Live Cell Metabolic Labeling and Imaging

et al. 2023

Self Cite

View full text Add to dashboard Cite

show abstract

“…Cell fixation and washing resulted in the loss of fluorescence from the cytoplasm, yet DNA labeling was maintained in cells that had incorporated TAMRA-dATP during S-phase (Figure 2c). 44 These results demonstrate that cells in culture can survive PCI and furthermore provide the first example of PCI release of metabolically functional building blocks of cellular biomolecules.…”

Section: ■ Results and Discussionmentioning

confidence: 75%

Active Uptake and Trafficking of Nucleoside Triphosphates In Vivo

et al. 2022

View full text Add to dashboard Cite

Modified nucleoside triphosphates (NTPs) are powerful probes and medicines, but their anionic character impedes membrane permeability. As such, invasive delivery techniques, transport carriers, or prodrug strategies are required for their in vivo use. Here, we present a fluorescent 2′-deoxyribonucleoside triphosphate “TAMRA-dATP” that exhibits surprisingly high bioavailability in vivo. TAMRA-dATP spontaneously forms nanoparticles in Mg+2-containing buffers that are taken into the vesicles of living cells and animals by energy-dependent processes. In cell cultures, photochemical activation with yellow laser light (561 nm) facilitated endosomal escape of TAMRA-dATP, resulting in its metabolic incorporation into DNA in vitro. In contrast, in vivo studies revealed that TAMRA-dATP is extensively trafficked by active pathways into cellular DNA of zebrafish (Danio rerio) and Caenorhabditis elegans where DNA labeling was observed in live animals, even without photochemical release. Metabolic labeling of DNA in whole, living animals can therefore be achieved by simply soaking animals in a buffer containing TAMRA-dATP or a structurally related compound, Cy3-dATP.

show abstract

“…We managed to remove partially the background fluorescence by washing with 50% DMF. 38 Thus, the arylethynyltrifluoroborate dienophile, tetrazine, and the IEDDA conditions we developed are compatible with proteins so they can be used for immunostaining on fixed cells.…”

Section: Resultsmentioning

confidence: 99%

Arylethynyltrifluoroborate Dienophiles for on Demand Activation of IEDDA Reactions

et al. 2021

Self Cite

View full text Add to dashboard Cite

Strained alkenes and alkynes are the predominant dienophiles used in inverse electron demand Diels–Alder (IEDDA) reactions. However, their instability, cross-reactivity, and accessibility are problematic. Unstrained dienophiles, although physiologically stable and synthetically accessible, react with tetrazines significantly slower relative to strained variants. Here we report the development of potassium arylethynyltrifluoroborates as unstrained dienophiles for fast, chemically triggered IEDDA reactions. By varying the substituents on the tetrazine (e.g., pyridyl- to benzyl-substituents), cycloaddition kinetics can vary from fast ( k 2 = 21 M –1 s –1 ) to no reaction with an alkyne-BF 3 dienophile. The reported system was applied to protein labeling both in the test tube and fixed cells and even enabled mutually orthogonal labeling of two distinct proteins.

show abstract

Flow cytometric determination of cell cycle progression via direct labeling of replicated DNA

Cited by 8 publications

References 20 publications

trans-Cyclooctene- and Bicyclononyne-Linked Nucleotides for Click Modification of DNA with Fluorogenic Tetrazines and Live Cell Metabolic Labeling and Imaging

trans-Cyclooctene- and Bicyclononyne-Linked Nucleotides for Click Modification of DNA with Fluorogenic Tetrazines and Live Cell Metabolic Labeling and Imaging

Active Uptake and Trafficking of Nucleoside Triphosphates In Vivo

Arylethynyltrifluoroborate Dienophiles for on Demand Activation of IEDDA Reactions

Contact Info

Product

Resources

About