Related Experiment Videos
Partial protection of oncogene, anti-sense oligodeoxynucleotides against serum nuclease degradation using terminal
1Cancer Research Campaign Department of Radiation Oncology, University of Liverpool, Clatterbridge Hospital, Bebington, Wirral, Merseyside, UK.
Abstract:
Under certain circumstances sequence-specific inhibition of gene expression may be achieved in intact cells using exogenous anti-sense oligodeoxynucleotides. The efficacy of this approach to investigating gene function is limited in part by the rapid serum nuclease mediated degradation of oligodeoxynucleotides in culture media. In order to determine the relative contributions of 3'-exonuclease, 5'-exonuclease and endonuclease activity in fetal calf serum to oligodeoxynucleotide destruction, we have tested chimeric N-ras anti-sense sequence molecules protected against exonuclease attack with terminal methylphosphonate diester linkages. An 18-mer with two methylphosphonate diester linkages at the 3'-terminus, a 20-mer with two methylphosphonate diester groups at both ends, and the 16-mer 3'-methylphosphonate monoester components of their respective piperidine hydrolysates were totally resistant to venom phosphodiesterase, whereas the 16-mer 3'-hydroxyl components of the hydrolysates were rapidly degraded. Both the chimeric oligodeoxynucleotides and 3'-methylphosphonate monoesters were considerably more stable than normal 3'-hydroxyl oligodeoxynucleotides at 37 degrees C in McCoy's 5A medium containing 15% heat inactivated fetal calf serum. Typically 20-30% of the former (initial concentration 10-100 microM) remained intact at 20 h as compared to the latter which were 88-100% degraded in 4 h and undetectable at 20 h. We conclude that a 3'-phosphodiesterase activity is a predominant nuclease responsible for oligodeoxynucleotide degradation by fetal calf serum, and that for cell culture studies, significant protection of oligodeoxynucleotides may be achieved by incorporating 3'-terminal methylphosphonate diester or even monoester end groups.
Insights
Oligodeoxynucleotides (ODNs) degrade quickly in cell culture media due to serum nucleases. Protecting ODNs with methylphosphonate linkages at the 3' end significantly enhances their stability, improving their use in gene function studies.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Gene expression can be inhibited using anti-sense oligodeoxynucleotides (ODNs).
- The use of ODNs is limited by their rapid degradation in cell culture media by serum nucleases.
- Understanding the specific nucleases involved is crucial for improving ODN stability.
Purpose of the Study:
- To identify the primary nucleases responsible for oligodeoxynucleotide degradation in fetal calf serum.
- To evaluate the protective effect of methylphosphonate linkages against nuclease degradation.
- To assess the stability of modified ODNs for potential use in cell culture studies.
Main Methods:
- Synthesized chimeric N-ras anti-sense oligodeoxynucleotides with terminal methylphosphonate diester linkages.
- Tested resistance of modified ODNs and their hydrolysates to venom phosphodiesterase.
- Assessed the stability of chimeric ODNs and methylphosphonate monoesters in cell culture medium (McCoy's 5A with 15% fetal calf serum) at 37°C over 20 hours.
Main Results:
- Methylphosphonate-protected ODNs and monoesters were resistant to venom phosphodiesterase, unlike 3'-hydroxyl components.
- Chimeric ODNs and 3'-methylphosphonate monoesters showed significantly enhanced stability in fetal calf serum compared to standard ODNs.
- At 20 hours, 20-30% of modified ODNs remained intact, while standard ODNs degraded by 88-100% within 4 hours.
Conclusions:
- 3'-phosphodiesterase activity is the predominant nuclease responsible for ODN degradation by fetal calf serum.
- Incorporating 3'-terminal methylphosphonate diester or monoester groups offers significant protection to ODNs.
- These modifications can improve the efficacy of ODNs for gene function investigations in cell culture.