Targeted elimination of zygotic messages in Xenopus laevis embryos by modified oligonucleotides possessing terminal cationic linkages

Abstract

We have designed a new class of modified antisense oligodeoxyribonucleotides (ODN) consisting of a central contiguous stretch of 6-8 unmodified nucleotides flanked by 3'- and 5'-regions containing several nucleotides joined by cationic internucleoside linkages. The positive charge results from modification of the internucleoside linkages as N, N -diethylethylene-diamine phosphoramidates. These zwitterionic compounds show improved antisense activity in both Xenopus oocytes and embryos compared to our previously described chimeric oligonucleotides possessing neutral terminal internucleoside linkages. Using the localized maternal mRNA An2 as a target, we have shown that chimeric oligonucleotides with terminal positive charges are very effective in the sequence-specific elimination of maternal messages present in both oocytes and embryos. In addition, using the embryonic mRNA GS17 as a target, we have shown that these oligonucleotides can direct RNase H-mediated cleavage of messages produced at the onset of zygotic transcription, after the mid-blastula stage. These new compounds should be useful in attenuating embryonic gene expression to study the role of specific proteins in early vertebrate development.

Figure 1

ODN sequences. ODNs 8N, 6N and 6P are complementary to the initiation codon region of the maternal Xenopus mRNA An2. ODNs GS8 and GS6 are complementary to the initiation codon region of the zygotic Xenopus mRNA GS17. ODNs sGS8 and sGS6 possess the same nucleotide composition, but the sequence is scrambled to differ from that of the GS17 ODNs. Asterisks (*) represent MEA internucleoside linkages, while plus signs (+) represent DEED-modified linkages. The structure of both modified linkages are shown below the ODN sequences.

Figure 2

Antisense activity of DEED-modified ODNs in oocytes. Mature X.laevis oocytes were injected with 12 ng of either 6P, 6N, 6N′, 8N or water (W). After 3 h, 10 oocytes were harvested and levels of An2 and An3 (unrelated control) mRNA were determined by northern analysis. NI, non-injected.

Figure 3

Antisense activity of DEED-modified ODNs in embryos. Single-cell X.laevis embryos were injected with 2 ng of ODN 6P or 6N. After 30 min, 10 embryos were harvested and levels of An2 (target) and histone H4 (control) mRNAs, as well as 18S rRNA, were determined by northern analysis. NI, non-injected.

Figure 4

Specific depletion of a zygotic transcript using DEED-modified ODNs. Single-cell X.laevis embryos were injected with either water (W) or 2 ng of ODN GS6 (AS), GS8 (AS), sGS6 (S) or sGS8 (S). After 10 h, 10 embryos were harvested and levels of GS17 (target) and histone H4 (control) mRNAs, as well as 18S rRNA, were determined by northern analysis. NI, non-injected.

Figure 5

Decreased GS17 mRNA levels are not a result of developmental arrest. Single-cell X.laevis embryos were injected with either water (W) or 2 ng of ODN GS6 or GS8. After 10–10.5 h, embryos were separated into groups possessing blastopore lips (+) and not possessing blastopore lips (–). Total RNA was isolated and levels of GS17 mRNA and 18S rRNA were determined by northern analysis. Ct, control ODN; NI, non-injected.