A. Rait et al., Inhibition of Ras p21 synthesis by antisense undecamers with uniform and specifically arranged phosphorothioate linkages, ANTI-CANC D, 11(3), 2000, pp. 181-191
The design of chimeric oligadeoxynucleotides (ODNs) in which certain phosph
odiester linkages are replaced by phosphorothioate (PS) aims to decrease no
n-sequence-specific effects of uniform PS ODNs and to preserve the PS-provi
ded protection against exo- and endonucleases. This study has, for the fist
time, directly compared the differences in nuclease resistance, cellular u
ptake, antisense potency and sequence specificity of PS and end-capped, pyr
imidine-protected (PPS) undecamer ODNs, that are complementary to the initi
ation codon region of human Hares mRNA. At concentrations above 5 mu M, bot
h PS and PPS undecamers were moderately and equally stable for over 48 h in
complete medium with RS485 cells overexpressing Ha-ras, They were complete
ly stable at 0.4 mu M when complexed with Lipofectin reagent that enhanced
cellular uptake up to 9-fold, Both the antisense PPS and PS undecamers prod
uced well-defined inhibition of Res p21 synthesis in both cell-free and cel
l-based assays. However, non-sequence-specific effects of the uniform phosp
horothioates were still significant. In contrast, the antisense PPS undecam
er, when delivered to RS485 cells with Lipofectin reagent, inhibits human R
as p21 synthesis by more than 90% at a concentration of 3.2 mu M, while the
effect of controls with inverted, mismatched or scrambled sequence was min
imal (5% or less) on p21 synthesis and RS485 cell growth, [(C) 2000 Lippinc
ott Williams & Wilkins.].