Comparison of Cellular Binding and Uptake of Antisense Phosphodiester, Phosphorothioate, and Mixed Phosphorothioate and Methylphosphonate Oligonucleotides

Abstract

The effects of phosphorothioate (S-oligonucleotide) or terminal phosphorothioate-phosphodiester (S-O-oligonucleotides) or methylphosphonate-phosphodiester (MP-O-oligonucleotides) modifications on mouse spleen cell surface binding, uptake, and degradation were studied using fluorescein (FITC)-conjugated oligonucleotides. S-oligonucleotides had the highest cell binding and uptake, followed by S-O-, O-, and MP-O-oligonucleotides. Competition studies indicated that S-oligonucleotides have an increased affinity for cell membrane oligonucleotide binding sites, because they could completely block O-oligonucleotide binding at a molar ratio of just 0.1. Uptake of all oligonucleotides was higher in B cells than T cells and was increased by stimulation with the B-cell mitogen, lipopolysaccharide. Although our cells had been purified using conventional techniques to eliminate dead cells, there remained about 5% of cells that were dead or dying, as determined by flow cytometry using propidium iodide staining. Of note, oligonucleotide association with dead cells was approximately 50-fold greater than that with living cells. Confocal microscopy confirmed that the oligonucleotides in living cells were intracellular, and indicated little nuclear uptake by 4 h. While extensive degradation of intracellular O-oligonucleotides was apparent by 4 h, there was no detectable degradation of S-, S-O, or MP-O-oligonucleotides.

Bacterial DNA activates human neutrophils by a CpG‐independent pathway

“…The presence of unmethylated CpG motifs, however, appears unnecessary for stimulation of neutrophil functions, as a similar degree of activation was observed using S-ODN containing or lacking these motifs. Although the half-life of P-ODN in culture is estimated at 4 h, while that of S-ODN is 14 h [37,38], it seems unlikely that differences in the stimulatory potential of the two classes of ODN could be attributed to a higher rate of P-ODN degradation; we assessed responses within a 30 min incubation period. Our observations that S-ODN stimulates neutrophil functions are also in agreement with Bylund et al [36], who found that S-ODN induce neutrophil NADPH-oxidase activation, leading to the release of reactive oxygen species, degranulation, and L-selectin shedding in a CpG-independent but phosphorothioatedependent manner.…”

Oligonucleotides with novel, cationic backbone substituents: aminoethylphosphonates

“…This technique has been demonstrated to be useful for studying and comparing the uptake and intracellular localization of different oligonucleotides conjugated to fluorescent molecules such as fluorescein [26]. Oligonucleotide uptake in cell lines has previously been shown to be saturable, sequence independent, and temperature and energy dependent [27].…”

Coadministration of antigen-conjugated and free CpG: Effects of in vitro and in vivo interactions in a murine model

“…Most of the researchers have focused on the cellular activating effect of the signal transduction events that are triggered by TLR9 ligation while less is known about the modulation of antigen uptake by CpG ODN. Some authors mentioned the relevance of 'stickiness' of synthetic ODNs [6,7] that can take part in the uptake of antigen and thus have an impact on the elicited immune response. There is evidence that ligation of CpG to a particulate antigen can enhance the uptake of CpG via endocytosis initiated by the antigen-specific BCR [8].…”