Synthesis and evaluation of nuclear targeting peptide-antisense oligodeoxynucleotide conjugates.

An endogenous nuclear enzyme, RNase H, is an important component in determining the efficacy of antisense oligodeoxynucleotides (ODNs). In an effort to improve the potency of antisense ODNs, conjugates with three different nuclear targeting signal peptides were prepared. These short peptide sequences have been shown to facilitate transport of macromolecules into the nucleus of cells. Efficient chemistry for the synthesis of ODN-peptide conjugates is described. Reaction of 5'-aminohexyl-modified ODNs with iodoacetic anhydride gave pure iodoacetamide ODNs (IA-ODNs) in good yield. These electrophilic intermediates were reacted with thiol-containing peptides to give ODN-peptides in excellent yield and purity. The ODN-peptides were further characterized by proteolysis with trypsin. Thermal denaturation studies with ssDNA targets showed little effect of the 5'-peptide modifications on the hybridization properties of the ODN. The effect of the nuclear signal peptides on antisense potency was evaluated in the freshwater ciliate Paramecium. A 3'-hexanol-modified 24-mer antisense ODN, complementary to the mRNA for calmodulin, alters regulation of membrane ion channels and swimming behavior of these cells. A 2'-O-methyl analog of this ODN was inactive, thus providing evidence that this activity in Paramecium is mediated by RNase H. Antisense ODN-nuclear signal peptide conjugates were transfected into the cells by electroporation. Surprisingly, these conjugates showed no antisense effects in comparison to a 5'-unmodified control ODN. Random peptides or amino acids conjugated to the 5'-terminus did not decrease antisense activity.