Comparison of cellular binding and uptake of antisense phosphodiester, phosphorothioate, and mixed phosphorothioate and methylphosphonate oligonucleotides.
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.
Department of Internal Medicine, University of Iowa College of Medicine, Iowa City.
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Zhao
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Matson
C J
CJ
Herrera
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Fisher
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Yu
A M
AM
Krieg
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D051379:Mice; D008811:Mice, Inbred DBA; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000493:pharmacokinetics*; D010755:Organophosphates / Q000493:pharmacokinetics; D009943:Organophosphorus Compounds / Q000493:pharmacokinetics*; D013154:Spleen / Q000166:cytology / Q000378:metabolism; D013873:Thionucleotides / Q000493:pharmacokinetics*
D016376:Oligonucleotides, Antisense; D010755:Organophosphates; D009943:Organophosphorus Compounds; D013873:Thionucleotides; C032627:methylphosphonic acid