Phosphodiester oligodeoxynucleotides bearing a 5' cholesteryl (chol) modification bind to low density lipoprotein (LDL), apparently by partitioning the chol-modified oligonucleotides into the lipid layer. Both HL60 cells and primary mouse spleen T and B cells incubated with fluorescently labeled chol-modified oligonucleotide showed substantially increased cellular association by flow cytometry and increased internalization by confocal microscopy compared to an identical molecule not bearing the chol group. Cellular internalization of chol-modified oligonucleotide occurred at least partially through the LDL receptor; it was increased in mouse spleen cells by cell culture in lipoprotein-deficient medium and/or lovastatin, and it was decreased by culture in high serum medium. To determine whether chol-modified oligonucleotides are more potent antisense agents, we titered antisense unmodified phosphodiester and chol-modified oligonucleotides targeted against a mouse immunosuppressive protein. Murine spleen cells cultured with 20 microM phosphodiester antisense oligonucleotides had a 2-fold increase in RNA synthesis, indicating the expected lymphocyte activation. Antisense chol-modified oligonucleotides showed an 8-fold increase in relative potency: they caused a 2-fold increase in RNA synthesis at just 2.5 microM. The increased efficacy was blocked by heparin and was further increased by cell culture in 1% (vs. 10%) fetal bovine serum, suggesting that the effect may, at least in part, be mediated via the LDL receptor. Antisense chol-modified oligonucleotides are sequence specific and have increased potency as compared to unmodified oligonucleotides.
We have evaluated FAST slides, a glass slide with a microporous polymeric surface that is a suitable substrate for microarray technology. The surface is a nitrocellulose-based polymer that binds DNA and proteins in a noncovalent but irreversible manner. FAST slides are compatible with robotic systems currently used to create microarrays and can easily accommodate volumes of 0.03-2 nL/spot. Our data indicate that FAST slides have a much higher binding capacity for DNA and better spot-to-spot consistency than traditional poly-lysine-coated slides. In addition, FAST slides are well suited for fluorescent detection because of their relatively low light scatter and efficient retention of arrayed DNA. These properties translate into fluorescent sensitivity comparable to modified glass surfaces. FAST slides are also ideal for arraying proteins, making them the only substrate of their kind currently available for microarray applications.
We have examined the cellular association and internalization of phosphodiester (PO) oligodeoxynucleotides (oligos) with HL60 cells. At 4 degrees C, a 15-mer PO homopolymer of thymidine (FOdT15) exhibits apparent saturation binding (Km = 22 +/- 1 nM) that is competitive with the binding of phosphorothioate (PS) oligos. The value of Kc for SdC28, a PS 28-mer homopolymer of cytidine, is 5 +/- 2 nM. SdC28 was used to strip cell surface fluorescence: Internalized fluorescence accumulated in a (concentration)(time)-dependent fashion, consistent with a pinocytotic mechanism. PS, and to a lesser extent, PO oligos inhibited the rate of internalization of fluorescent albumin, also a marker of pinocytosis. This was correlated with direct in vitro inhibition of protein kinase C (PKC) beta 1 by the PS and PO oligos. Furthermore, other PKC inhibitors (H7, staurosporine, DMSO, PKC pseudosubstrate polypeptide) also inhibited intracellular accumulation of pinocytosed materials, perhaps by stimulating the exocytosis rate. In HL60 cells, the pinocytotic internalization of charged oligos appears to be dependent on intact PKC kinase activity, which is inhibited in vitro by PS and PO oligos.
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