Skip to main content
SpringerLink
Log in

Synthesis of photolabile dUTP analogues and their enzymatic incorporation for DNA labeling

  • Articles
  • Special Issue · The Frontiers of Chemical Biology and Synthesis
  • Published:
Science China Chemistry Aims and scope Submit manuscript

Abstract

Two new photolabile nucleotide analogues with furan-fused deoxyuridine were synthesized through Sonogashira coupling. Their enzymatic incorporation into DNA was evaluated with two DNA polymerases (Taq and Deep vent exo-) by polymerase chain reaction (PCR). Deep vent exo-recognized both nucleotides as substrates for primer extension, while Taq was much less proficient. Light irradiation of PCR products released the amino and carboxyl moieties of DNA. Further labeling with fluorescein isothiocyanate for a long DNA construct with F-dUnTP incorporation was successfully achieved.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Greene TW, Wuts PGM, editors. Protective Groups in Ogranic Synthesis. New York: John Wiley & Sons Inc., 1999

    Google Scholar 

  2. Kahl JD, McMinn DL, Greenberg MM. High-yielding method for on-column derivatization of protected oligodeoxy-nucleotides and its application to the convergent synthesis of 5′,3′-bis-conjugates. J Org Chem, 1998, 63: 4870–4871

    Article  CAS  Google Scholar 

  3. Kahl JD, Greenberg MM. Introducing structural diversity in oligonucleotides via photolabile, convertible c5-substituted nucleotides. J Am Chem Soc, 1999, 121: 597–604

    Article  CAS  Google Scholar 

  4. Burley GA, Gierlich J, Mofid MR, Nir H, Tal S, Eichen Y, Carell T. Directed DNA metallization. J Am Chem Soc, 2006, 128: 1398–1399

    Article  CAS  Google Scholar 

  5. Giller G, Tasara T, Angerer B, M U Hlegger K, Amacker M, Winter H. Incorporation of reporter molecule-labeled nucleotides by DNA polymerases. I. Chemical synthesis of various reporter group-labeled 2-deoxyribonucleoside-5-triphosphates. Nucleic Acids Res, 2003, 10: 2630–2635

    Article  Google Scholar 

  6. Niedringhaus TP, Milanova D, Kerby MB, Snyder MP, Barron AE. Landscape of next-generation sequencing technologies. Anal Chem, 2011, 83: 4327–4341

    Article  CAS  Google Scholar 

  7. Rosi NL, Mirkin CA. Nanostructures in biodiagnostics. Chem Rev, 2005, 105: 1547–1562

    Article  CAS  Google Scholar 

  8. Sassolas A, Leca-Bouvier BD, Blum LJ. DNA biosensors and microarrays. Chem Rev, 2008, 108: 109–139

    Article  CAS  Google Scholar 

  9. Venkatesan N, Kim BH. Peptide conjugates of oligonucleotides: Synthesis and applications. Chem Rev, 2006, 106: 3712–3761

    Article  CAS  Google Scholar 

  10. Hocek M, Fojta M. Nucleobase modification as redox DNA labelling for electrochemical detection. Chem Soc Rev, 2011, 40: 5802–5814

    Article  CAS  Google Scholar 

  11. Bergen K, Steck AL, Strutt S, Baccaro A, Welte W, Diederichs K, Marx A. Structures of klentaq DNA polymerase caught while incorporating c5-modified pyrimidine and c7-modified 7-deazapurine nucleoside triphosphates. J Am Chem Soc, 2012, 134: 11840–11843

    Article  CAS  Google Scholar 

  12. Borsenberger V, Kukwikila M, Howorka S. Synthesis and enzymatic incorporation of modified deoxyuridine triphosphates. Org Biomol Chem, 2009, 7: 3826–3835

    Article  CAS  Google Scholar 

  13. Seela F, Sirivolu VR, Chittepu P. Modification of DNA with octadiynyl side chains synthesis, base pairing, and formation of fluorescent coumarin dye conjugates of four nucleobases by the alkyne-azide “click” reaction. Bioconjugate Chem, 2007, 1: 211–224

    Google Scholar 

  14. Weisbrod SH, Marx A. A nucleoside triphosphate for site-specific labelling of DNA by the staudinger ligation. Chem Commun, 2007: 1828-1830

  15. Augustin MA, Ankenbauer W, Angerer B. Progress towards single-molecule sequencing: Enzymatic synthesis of nucleotide-specifically labeled DNA. J Biotechnol, 2001, 86: 289–301

    Article  CAS  Google Scholar 

  16. Raindlova V, Pohl R, Hocek M. Synthesis of aldehyde-linked nucleotides and DNA and their bioconjugations with lysine and peptides through reductive amination. Chem Eur J, 2012, 18: 4080–4087

    Article  CAS  Google Scholar 

  17. Seo TS, Bai X, Kim DH, Meng Q, Shi S, Ruparel H, Li Z, Turro NJ, Ju J. Four-color DNA sequencing by synthesis on a chip using photocleavable fluorescent nucleotides. Proc Nat Acad Sci USA, 2005, 102: 5926–5931

    Article  CAS  Google Scholar 

  18. Wu W, Stupi BP, Litosh VA, Mansouri D, Farley D, Morris S, Metzker S, Metzker ML. Termination of DNA synthesis by n6-alkylated, not 3′-O-alkylated, photocleavable 2′-deoxyadenosine triphosphates. Nucleic Acids Res, 2007, 35: 6339–6349

    Article  CAS  Google Scholar 

  19. Litosh VA, Wu W, Stupi BP, Wang J, Morris SE, Hersh MN, Metzker ML. Improved nucleotide selectivity and termination of 3′-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzyl alkylated homedu triphosphates. Nucleic Acids Res, 2011, 39: e39

    Article  CAS  Google Scholar 

  20. Eid J, Fehr A, Gray J, Luong K, Lyle J, Otto G, Peluso P, Rank D, Wu D, Yang A, Zaccarin D, Zhao P, Zhong F, Korlach J, Turner S. Real-time DNA sequencing from single polymerase molecules. Science, 2009, 5910: 133–138

    Article  Google Scholar 

  21. Stupi BP, Li H, Wang J, Wu W, Morris SE, Litosh VA, Muniz J, Hersh MN, Metzker ML. Stereochemistry of benzylic carbon substitution coupled with ring modification of 2-nitrobenzyl groups as key determinants for fast-cleaving reversible terminators. Angew Chem Int Ed Engl, 2012, 51: 1724–1727

    Article  CAS  Google Scholar 

  22. Su M, Wang J, Tang X. Photocaging strategy for functionalisation of oligonucleotides and its applications for oligonucleotide labelling and cyclisation. Chem Eur J, 2012, 18: 9628–9637

    Article  CAS  Google Scholar 

  23. Sonogashira K, Tohda Y, Hagihara N. A convenient synthesis of acetylenes: Catalytic substitutions of acetylenic hydrogen with bromoalkenes, iodoarenes and bromopyridines. Tetrahedron Lett, 1975, 50: 4467–4470

    Article  Google Scholar 

  24. Burgess K, Cook D. Syntheses of nucleoside triphosphates. Chem Rev, 2000, 2: 2047–2060

    Article  Google Scholar 

  25. Kovács T, Ötvös L. Simple synthesis of 5-vinyl- and 5-ethynyl-2′-deoxyuridine-5′-triphosphates. Tetrahedron Lett, 1988, 36: 4525–4528

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. State Key Laboratory of Natural and Biomimetic Drugs; the School of Pharmaceutical Sciences, Peking University, Beijing, 100191, China

    JunZhou Wu, Jie Wang & XinJing Tang

  2. State key Laboratory of Drug Research; Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China

    XinJing Tang

Authors
  1. JunZhou Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jie Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. XinJing Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to XinJing Tang.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wu, J., Wang, J. & Tang, X. Synthesis of photolabile dUTP analogues and their enzymatic incorporation for DNA labeling. Sci. China Chem. 57, 322–328 (2014). https://doi.org/10.1007/s11426-013-5034-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11426-013-5034-2

Keywords

Search

Navigation