Sa. Ciafre et al., STABILITY AND FUNCTIONAL EFFECTIVENESS OF PHOSPHOROTHIOATE MODIFIED DUPLEX DNA AND SYNTHETIC MINI-GENES, Nucleic acids research, 23(20), 1995, pp. 4134-4142
Several gene transfer techniques that employ 'naked DNA' molecules hav
e recently been developed and numerous gene therapy protocols that mak
e use of 'naked-DNA' have been proposed. We studied the possibility of
enhancing the stability of 'naked DNA vectors' and thus also gene tra
nsfer and expression efficiencies, by constructing phosphorothioate (P
S-) double strand DNA molecules and functional transcription units. We
first synthesized short PS-double strand DNA molecules by the anneali
ng of two complementary, 35 nt long, oligonucleotides. The accessibili
ty of DNA modifying enzymes to this molecule was significantly decreas
ed: T4-ligase and kinase activity were respectively reduced up to 1/2
and to 1/6, as compared to the normal phosphodiester molecule. Nucleol
ytic stability was increased either to purified enzymes (DNase I and B
aI31) or to incubations in fresh serum, cell culture medium or in musc
le protein extract. Phosphorothioate end-capped complete eukaryotic tr
anscription units (obtained by Tao polymerase amplification with PS-pr
imers) were not significantly protected from nucleolytic attack. On th
e contrary, synthetic transcription units, 'mini genes', obtained by T
ag amplification with 1, 2 or 3 PS-dNTP substitutions, were resistant
to DNase I and BaI31 nucleolytic activity. Transcription efficiency, d
riven by the T7 promoter, was 96.5, 95 and 33.5%(respectively with 1,
2 or 3 substitutions), as compared to the normal phosphodiester molecu
les.