Mw. Reed et al., SYNTHESIS AND EVALUATION OF NUCLEAR TARGETING PEPTIDE ANTISENSE OLIGODEOXYNUCLEOTIDE CONJUGATES, Bioconjugate chemistry, 6(1), 1995, pp. 101-108
endogenous nuclear enzyme, RNase H, is an important component in deter
mining the efficacy of antisense oligodeoxynucleotides (ODNs). In an e
ffort 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 macromol
ecules into the nucleus of cells. Efficient chemistry for the synthesi
s of ODN-peptide conjugates is described. Reaction of 5'-aminohexyl-mo
dified ODNs with iodoacetic anhydride gave pure iodoacetamide ODNs (IA
-ODNs) in good yield. These electrophilic intermediates were reacted w
ith 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 pro
perties of the ODN. The effect of the nuclear signal peptides on antis
ense potency was evaluated in the freshwater ciliate Paramecium. A 3'-
hexanol-modified 24-mer antisense ODN, complementary to the mRNA for c
almodulin, alters regulation of membrane ion channels and swimming beh
avior of these cells. A 2'-O-methyl analog of this ODN was inactive, t
hus providing evidence that this activity in Paramecium is mediated by
RNase H. Antisense ODN-nuclear signal peptide conjugates were transfe
cted 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.