Nucleic Acids Research, Vol 26, Issue 7 1567-1575, Copyright © 1998 by Oxford University Press
RV Giles, DG Spiller, J Grzybowski, RE Clark, P Nicklin and DM Tidd
It is widely accepted that most cell types efficiently exclude
oligonucleotides in vitro and require specific delivery systems, such as
cationic lipids, to enhance uptake and subsequent antisense effects.
Oligonucleotides are not readily transfected into leukaemia cell lines
using cationic lipid systems and streptolysin O (SLO) is used to effect
their delivery. We wished to investigate the optimal oligonucleotide
composition for antisense efficacy and specificity following delivery into
leukaemia cells using SLO. For this study the well characterised chronic
myeloid leukaemia cell line KYO-1 was selected and oligonucleotides
(20mers) were targeted to an empirically identified accessible site of c-
myc mRNA. The efficiency and specificity of antisense effect was measured 4
and 24 h after SLO-mediated delivery of the oligonucleotides. C5-propyne
phosphodiester and phosphorothioate compounds were found to present
substantial non-specific effects at 20 microM but were inactive at 0.2
microM. Indeed, no antisense-specific effect was noted at any concentration
at either time. All of the other oligonucleotides tested induced some
measurable antisense effect, except 7 (chimeric, all-phosphorothioate,
2'-methoxyethoxy termini) which was essentially inactive at 20 microM. The
rank efficiency order of the remaining antisense compounds was 4 = 3
>> 9 >> 10 = 8 = 5 = 6 > 11. The efficient antisense effects
induced by the chimeric methylphosphonate-phosphodiester compounds were
found to be highly specific. Increased phosphorothioate content in the
oligonucleotide backbone correlated with reduced antisense activity
(efficacy: 2'- methoxyethoxy series 9 >> 8 >> 7,
2'-methoxytriethoxy series 10 > 11). No consistent evidence was obtained
for increased activity correlating with increased oligonucleotide-mRNA
heteroduplex thermal stability. In conclusion, the chimeric
methylphosphonate-phosphodiester oligodeoxynucleotides present the most
favourable characteristics of the compounds tested, for efficient and
specific antisense suppression of gene expression following SLO-mediated
delivery.
ARTICLES
Selecting optimal oligonucleotide composition for maximal antisense effect following streptolysin O-mediated delivery into human leukaemia cells
School of Biological Sciences, University of Liverpool, Life Science Building, Crown Street, Liverpool L69 7ZB, UK. giles@liv.ac.uk
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