Comparison of binding of N3'-->P5' phosphoramidate and phosphorothioate oligonucleotides to cell surface proteins of cultured cells.
The binding of uniformly modified N3'-->P5' phosphoramidate and stereorandom and stereopure phosphorothioate oligonucleotides (ODN) to cell surface proteins was studied, using both a fibroblast and an epithelial cell line, to assess the effect of different analog backbone types and base composition on cell surface protein binding. Marked differences were observed, both quantitative and qualitative, in the proteins to which individual ODN bound. One phosphoramidate, antisense to the insulin-like growth factor-1 (IGF-1) receptor (IGF-1R), bound to different proteins than did either a 6-base mismatch phosphoramidate IGF-1R sequence or a sense N-ras sequence. The latter bound poorly to the fibroblast line and predominantly to a 46 kDa protein in the epithelial line, as did many of the other ODN. This binding was not so marked as that of the isosequential end-capped phosphodiester N-ras sequence, which bound to this protein in both cell lines. Stereopure and stereorandom phosphorothioates containing a G-quartet (shown in other studies to form high-order tetrad structures), antisense to c-myc, exhibited considerable nonspecific binding to many proteins, as did the isosequential phosphoramidate. In particular, this ODN sequence gave notable binding to high molecular weight proteins. In general, binding of the c-myc ODN to proteins of 28-30, 46, 67, and 70-90 kDa was found in this study.
Hawley
P
P
University of East Anglia, Norwich, UK.
Nelson
J S
JS
Fearon
K L
KL
Zon
G
G
Gibson
I
I
D016475:3T3 Cells; D000818:Animals; D020137:Base Pair Mismatch; D001665:Binding Sites; D002289:Carcinoma, Non-Small-Cell Lung / Q000473:pathology; D002462:Cell Membrane / Q000378:metabolism; D016895:Culture Media, Serum-Free; D004591:Electrophoresis, Polyacrylamide Gel; D004847:Epithelial Cells / Q000378:metabolism; D008175:Lung Neoplasms; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015689:Oncogene Protein p21(ras) / Q000235:genetics; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D017526:Receptor, IGF Type 1 / Q000235:genetics; D013237:Stereoisomerism; D013873:Thionucleotides / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D016895:Culture Media, Serum-Free; D008565:Membrane Proteins; D016376:Oligonucleotides, Antisense; D009943:Organophosphorus Compounds; D016271:Proto-Oncogene Proteins c-myc; D013873:Thionucleotides; D017526:Receptor, IGF Type 1; D015689:Oncogene Protein p21(ras)