Improved Anti-Herpes Simplex Virus Type 1 Activity of a Phosphodiester Antisense Oligonucleotide Containing a 3′al Hairpin-Like Structure

We synthesized a series of 20-mer antisense phosphodiester oligonucleotides constituting of a 5′dodecameric sequence, complementary to the acceptor splice junction of herpes simplex virus type 1 (HSV-1) pre-mRNAs IE4 and IE5, flanked in 3′ by octameric sequences adopting hairpin-like structures of different stabilities. The presence of the minihairpins in 3′ protected the 20-mer phosphodiester oligonucleotides against serum nuclease degradation, this protection being well correlated to the reported melting temperatures of the minihairpins, and to the gel mobilities of the 20-mer oligonucleotides. While no protection was observed using a linear 8-mer, the addition in 3′ of the most stable minihairpin—H8—increased more than eightfold the nuclease resistance of the linear antisense dodecamer. We analyzed the effect of such a protection on the anti-HSV-1 antisense activities of the oligonucleotides. When bearing H8 in 3′, the antisense dodecamer was 10 times more active than in the absence of 3′-flanking sequence, while a linear 20-mer control containing the antisense sequence was only 3 times more active. This work provides the basis for a further rational design of phosphodiester antisense oligonucleotides, taking advantage of the specific properties conferred by their conformations.