Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Mar 27;34(4):772-780.
doi: 10.1021/acs.bioconjchem.3c00064. Online ahead of print.

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

Ambra Spampinato  1   2 , Erika Kužmová  1 , Radek Pohl  1 , Veronika Sýkorová  1 , Milan Vrábel  1 , Tomáš Kraus  1 , Michal Hocek  1   2
Affiliations

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

Ambra Spampinato et al. Bioconjug Chem. .

Abstract

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.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
(A) Click reaction of dCXTP with tetrazines T1-T3; (B) enzymatic synthesis of 19DNA_CX (X = 4TCO, 2TCO, p4TCO, p2TCO, pBCN) and the consequent click reaction of 19DNA_CX with tetrazines T1–T4 and magnetoseparation to obtain 19ON_CXTY; (C) PAGE analysis of the PEX reactions with modified dCX (X = 4TCO, 2TCO, p4TCO, p2TCO, pBCN) using KOD XL DNA polymerase, primer primA, and template Temp19_1C. P: primer, (C+): natural dGTP, (C−): natural dGTP without dCTP, (CTCO/BCN): dC4TCO or dC2TCOdCp4TCO or dCp2TCO or dCpBCN, dGTP; (D) denaturing PAGE analysis of the DNA-tetrazine reaction of 19DNA_C4TCO with T1 (5 μM) lane 3 or T1 (50 μM) lane 4; of 19DNA_C2TCO with T1 (5 μM) lane 6 or T1 (50 μM) lane 7; of 19DNA_Cp4TCO with T1 (5 μM) lane 9 or T1 (50 μM) lane 10; of 19DNA_Cp2TCO with T1 (5 μM) lane 12 or T1 (50 μM) lane 13; of 19DNA_CpBCN with T1 (5 μM) lane 15 or T1 (50 μM) lane 16. Negative controls (−): 19DNAnatural lane 1, 19DNA_C4TCO lane 2, 19DNA_C2TCO lane 5, 19DNA_Cp4TCO lane 8, 19DNA_Cp2TCO lane 11, 19DNA_CpBCN lane 14. (E) Fluorescence time-lapse measurements of dCp4TCOTP or dCp2TCOTP or dCpBCNTP with T1 tetrazine showing changes in the fluorescence signal of the click products in time (Supporting Information, Section S3.1, Figure S10C).
Scheme 1
Scheme 1. Synthesis of 4TCO-, 2TCO-, p4TCO-, p2TCO-, and pBCN-Modified 2′-Deoxycitidine Triphosphates
Conditions: (i) 2-TCO–NHS–carbonate, or 4-TCO–NHS–carbonate, or 2-TCO-PEG3-NHS-carbonate, or 4-TCO-PEG3-NHS-carbonate, or endo-BCN-PEG3-NHS-ester in H2O/TEAB 1 M, dimethylformamide, 55 °C, 4 h.
Figure 2
Figure 2
Modified dNTPs (dC2TCOTP, dC4TCOTP, dCp2TCOTP, dCp4TCOTP, or dCpBCNTP) were delivered to U-2 OS cells with the SNNT1 transporter and further incubated in the cultivation medium for 60 min. Then, the cells were incubated with tetrazines T1–T4 and imaged by confocal microscopy.
Figure 3
Figure 3
Confocal microscopy imaging of U-2 OS cells with labeled DNA. Cells were treated with dCp4TCOTP/SNTT1 (A–F; 10 μM) or dCpBCNTP/SNTT1 (G–I; 10 μM) for 5 min, then incubated in a medium for 60 min. Subsequently, tetrazine T1 (A–C; 1 μM; 15 min) or T3 (D–F; 10 μM; 4 h) was added. Cells incubated with dCpBCNTP (G–I) were fixed with methanol before tetrazine T4 (G–I; 2 μM; 30 min) was added. Prior to microscopy, DRAQ5 (B–C; 2.5 μM), or HOECHST 33342 (E–F; 3 μM), or DAPI (H–I; 0.5 μM) was added. T1 channel (A), DRAQ5 channel (B), and merged (C); T3 channel (D), HOECHST 33342 (E), and merged (F); T4 channel (G), DAPI (H), and merged (I). Bar size 50 μm.
Figure 4
Figure 4
DNA incorporation-based cell cycle analysis of U-2 OS cells. (A) The cells were pulse-treated with a mixture of dCpBCNTP/SNTT 1 (10 μM) in tricine buffer for 4 min and further incubated in a conditioned medium for 1 h. After this period, the cells were fixed with methanol, incubated with 2 μM TAMRA-tetrazine (T4) for 30 min at 37 °C, counterstained with DAPI, and analyzed by flow cytometry (B).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Ligasová A.; Koberna K. DNA Replication: From Radioisotopes to Click Chemistry. Molecules 2018, 23, 3007.10.3390/molecules23113007. - DOI - PMC - PubMed
    1. Gratzner H. G. Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: A new reagent for detection of DNA replication. Science 1982, 218, 474–475. 10.1126/science.7123245. - DOI - PubMed
    1. Salic A.; Mitchison T. J. A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 2415–2420. 10.1073/pnas.0712168105. - DOI - PMC - PubMed
    1. Zawada Z.; Tatar A.; Mocilac P.; Budesinsky M.; Kraus T. Transport of Nucleoside Triphosphates into Cells by Artificial Molecular Transporters. Angew. Chem., Int. Ed. 2018, 130, 10039–10043. 10.1002/ange.201801306. - DOI - PubMed
    1. Güixens-Gallardo P.; Zawada Z.; Matyašovský J.; Dziuba D.; Pohl R.; Kraus T.; Hocek M. Brightly Fluorescent 2′-Deoxyribonucleoside Triphosphates Bearing Methylated Bodipy Fluorophore for in Cellulo Incorporation to DNA, Imaging, and Flow Cytometry. Bioconjugate Chem. 2018, 29, 3906–3912. 10.1021/acs.bioconjchem.8b00721. - DOI - PubMed