Partial protection of oncogene, anti-sense oligodeoxynucleotides against serum nuclease degradation using terminal methylphosphonate groups

Dm Tidd,Hm Warenius

doi:10.1038/bjc.1989.283

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.

Highlights

Our results suggest that a 3'-phosphodiesterase activity plays a predominant role in the breakdown of oligodeoxynucleotides by fetal calf serum and that significant increases in the half lives of intact molecules in tissue culture medium can be achieved by merely protecting the 3' ends with methylphosphonate diester or even monoester groups
Syntheses of the c-myc 1 5-mer oligodeoxynucleotides described by Wickstrom et al (1986, 1988) and others (Heikkila et al, 1987; Holt et al, 1988; Harel-Bellan et al, 1988) were repeated as part of a project to determine the general applicability of the reported anti-sense approach to inhibiting myc gene expression in intact cells
A ladder of oligonucleotide degradation products was apparent, differing by the successive removal of a single nucleotide unit and decreasing in abundance with decreasing chain length. Such a pattern implicated a predominantly exonucleolytic attack by serum nucleases, and it seemed likely that a major factor in this would be the 3'phosphodiesterase activity we had previously encountered in our work on nucleotide prodrugs (Tidd et al, 1982)

Summary

Methods

Methylphosphonodiester/phosphodiester chimeric oligodeoxynucleotides were very kindly assembled for us on an automatic DNA synthesiser by Stephen Bates of Applied Biosystems, Warrington, Cheshire, and were synthesised in our own laboratory on an Applied Biosystems 381A Synthesizer, using a combination of methylphosphonamidite and f3-cyanoethyl phosphoramidite chemistries, developed from the original work of Dorman et al (1984). The oligonucleotides, in the fully protected, trityl-on, controlled pore glass support-bound form, were deprotected following the procedure for methylphosphonate oligonucleotide analogues (Maher & Dolnick, 1988). The products, carrying 5'-dimethoxytrityl groups as the sole remaining protecting functions, were dissolved in 5 ml 0.1 M. Human N-ras gene sequence met thr glu tyr lys leu val .... Components of 1M piperidine hydrolyzate of 5'-(P) (M) -3' 18-mer 5'-OH,3'-OH (P)

Results

Discussion

Conclusion