Characterisation of membrane oligonucleotide-binding proteins and oligonucleotide uptake in keratinocytes

Inadequate cellular compartmentalisation of plasmid DNA and antisense oligo deoxynucleotides (ODNs) is generally considered as a major limitation in th eir use. In this study, an approach combining in situ visualisation of rhod amine-labelled ODNs and affinity modification of proteins by radiolabelled- alkylating ODN derivatives has been used to investigate the uptake of ODNs into keratinocytes. We confirm here that unmodified ODNs are efficiently ta ken up and accumulate in cell nuclei in primary keratinocytes as well as in HaCaT and A431 keratinocyte cell lines. Uptake is fast, irreversible, satu rable and not significantly altered by incubation at low temperature. Affin ity modification studies in keratinocyte cell lines has revealed two high-a ffinity, cell-specific interactions between ODNs and proteins of 61-63 kDa and 35 kDa, Trypsin pre-treatment of A431 cells and pre-incubation with pol yanions, or with unlabelled nucleic acid competitors, inhibited the accumul ation of rhodamine-labelled ODNs in nuclei as well as the affinity labellin g of the 61-63 kDa doublet and 35 kDa ODN-binding proteins by reactive ODN derivatives. Finally, cell fractionation studies indicated that these ODN-b inding proteins were essentially localised in the plasma membrane. Our resu lts suggest that these ODN-binding proteins might be involved in the recogn ition and transport of ODNs into keratinocytes.