Skip to main content

Integration of Phosphodiesterase-Induced Degradation of Oligonucleotides with Capillary Polymer-Sieving Electrophoresis

  • Protocol
Capillary Electrophoresis of Nucleic Acids

Part of the book series: Methods in Molecular Biology ((MIMB,volume 162))

  • 601 Accesses

Abstract

Synthetic oligodeoxyribonucleotides (ODN) have been proposed as a class of potential therapeutic agents that can interact in a rational way with DNA or RNA, with the aim of inhibiting the expression of unwanted genetic information (13). One of the most critical questions in the evaluation of these molecules, is their stability toward enzymatic breakdown by [3′ or 5′]-exonucleases and endonucleases (4). In order to inhibit or at least limit the effect of these nucleases, chemically modified ODNs have been synthesized.

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

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Stein C. A. and Cheng Y.-C. (1993) Antisense oligonucleotides as therapeutic agents—is the bullet really magical? Science 261, 1004–1012.

    Article  PubMed  CAS  Google Scholar 

  2. Milligan J. F., Matteucci M. D., and Martin J. C. (1993) Current concepts in antisense drug design. J. Med. Chem. 36, 1923–1937.

    Article  PubMed  CAS  Google Scholar 

  3. De Mesmaeker A., Häner R., Martin P., and Moser H. E. (1995) Antisense oligonucleotides. Acc. Chem. Res. 28, 366–374.

    Article  Google Scholar 

  4. Stein C. A., Subasinghe C., Shinozuka K., and Cohen J. S. (1988) Physicochemical properties of phosphorothioate oligodeoxynucleotides. Nucleic Acids Res. 16, 3209–3221.

    Article  PubMed  CAS  Google Scholar 

  5. Khan K., Liekens K., Van Aerschot A., Van Schepdael A., and Hoogmartens J. (1997) Stability measurement of oligonucleotides in serum samples using capillary electrophoresis. J. Chromatogr. B 702, 69–76.

    Article  CAS  Google Scholar 

  6. Campbell J.M., Bacon T.A., and Wickstrom E. (1990) Oligonucleoside phosphorothioate stability in subcellular extracts, culture media, sera and cerebrospinal fluid J. Biochem. Biophys. Meth. 20, 259–267.

    Article  PubMed  CAS  Google Scholar 

  7. Eder P. S., DeVine R. J., Dagle J. M., and Walder J. A. (1991) Substrate specificity and kinetics of degradation of antisense oligonucleotides by a 3′ exonuclease in plasma. Antisense Res. Dev. 1, 141–151.

    PubMed  CAS  Google Scholar 

  8. Akhtar S., Kole R., and Juliano R. L. (1991) Stability of antisense DNA oligodeoxy-nucleotide analogs in cellular extracts and sera. Life Sciences 49, 1793–1801.

    Article  PubMed  CAS  Google Scholar 

  9. Shaw J.-P., Kent K., Bird J., Fishback J., and Froehler B. (1991) Modified deoxyoligonucleotides stable to exonuclease degradation in serum. Nucleic Acids Res. 19,747–750.

    Article  PubMed  CAS  Google Scholar 

  10. Guttman A. and Ulfelder K. J. (1998) Separation of DNA by capillary electrophoresis. Adv. Chromatogr. 38, 301–340.

    PubMed  CAS  Google Scholar 

  11. Bruin G. J. M., Börnsen K. O., Hüsken D., Gassmann E., Widmer H. M., and Paulus A. (1995) Stability measurements of antisense oligonucleotides by capillary gel electro-phoresis. J. Chromatogr. A 709, 181–195.

    Article  PubMed  CAS  Google Scholar 

  12. Shi X., Hammond R. W., and Morris M. D. (1995) DNA conformational dynamics in polymer solutions above and below the entanglement limit. Anal. Chem. 67, 1132–1138.

    Article  PubMed  CAS  Google Scholar 

  13. Heiger D. N., Cohen A. S., and Karger B. L. (1990) Separation of DNA restriction fragments by high performance capillary electrophoresis with low and zero crosslinked polyacrylamide using continuous and pulsed electric fields. J. Chromatogr. 516, 33–48.

    Article  PubMed  CAS  Google Scholar 

  14. Palm A. and Hjertén S. (1996) The resolution of DNA fragments in capillary electrophoresis in replaceable agarose gels. J. Capillary Electrophor. 3, 173–179.

    PubMed  CAS  Google Scholar 

  15. Fung E. N. and Yeung E. S. (1995) High speed DNA sequencing by using mixed poly(ethylene oxide) solutions in uncoated capillary columns. Anal. Chem. 67, 1913–1919.

    Article  CAS  Google Scholar 

  16. Cheng J., Kasuga T., Watson N. D., and Mitchelson K. R. (1995) Enhanced singlestranded DNA conformation polymorphism analysis by entangled solution capillary electrophoresis. J. Capillary Electrophor. 2, 24–29.

    CAS  Google Scholar 

  17. Khan K., Van Schepdael A., and Hoogmartens J. (1996) Capillary electrophoresis of oligonucleotides using replaceable sieving buffer with low viscosity-grade hydroxyethyl cellulose. J. Chromatogr. A 742, 267–274.

    Article  CAS  Google Scholar 

  18. Regnier F. E., Patterson D. H., and Harmon B. J. (1995) Electrophoretically-mediated microanalysis (EMMA). Trends Anal. Chem. 14, 177–181.

    CAS  Google Scholar 

  19. Bao J. and Regnier F. E. (1992) Ultramicro enzyme assays in a capillary electrophoretic system J. Chromatogr. 608, 217–224.

    Article  PubMed  CAS  Google Scholar 

  20. Cellosize® HEC EP09 safety data sheet. (1997) Union Carbide, Antwerp, Belgium.

    Google Scholar 

  21. Saevels J., Huygens K., Van Schepdael A., and Hoogmartens J. (1997) In-line coupling of the enzymatic degradation of oligonucleotides with capillary polymer sieving electrophoresis. Anal. Chem. 69, 3299–3303.

    Article  CAS  Google Scholar 

  22. Saevels J., Van Schepdael A., and Hoogmartens J. (1997) Phosphodiesterase susceptibility of modified oligonucleotides studied in an integrated capillary electrophoresis system. J. Capillary Electrophor. 4, 167–172.

    PubMed  CAS  Google Scholar 

  23. Björk W. (1963) Purification of phosphodiesterase from Bothrops atrox venom, with special consideration of the elimination of monophosphatases. J. Biol. Chem. 238, 2487–2490.

    PubMed  Google Scholar 

  24. Richards G. M., du Vair G., and Laskowski M. (1965) Comparison of the levels of phosphodiesterase, endonuclease, and monophosphatases in several snake venoms. Biochemistry 4, 501–503.

    Article  PubMed  CAS  Google Scholar 

  25. Vandendriessche F., Van Aerschot A., Voortmans M., Janssen G., Busson R., Van Overbeke A., et al. (1993) Synthesis, enzymatic stability and base-pairing properties of oligothymidylates containing thymidine dimers with different N-substituted guanidine linkages. J. Chem. Soc. Perkin. Trans. 1,1567–1575.

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2001 Humana Press Inc.

About this protocol

Cite this protocol

Saevels, J., Schepdael, A.V., Hoogmartens, J. (2001). Integration of Phosphodiesterase-Induced Degradation of Oligonucleotides with Capillary Polymer-Sieving Electrophoresis. In: Mitchelson, K.R., Cheng, J. (eds) Capillary Electrophoresis of Nucleic Acids. Methods in Molecular Biology, vol 162. Humana Press. https://doi.org/10.1385/1-59259-055-1:443

Download citation

  • DOI: https://doi.org/10.1385/1-59259-055-1:443

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-779-3

  • Online ISBN: 978-1-59259-055-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics