A hexanucleotide selected for increased cellular uptake in cis contains a highly active CpG-motif in human B cells and primary peripheral blood mononuclear cells.
The relationship between immunostimulation of human B cells by cytosine-phosphate-guanosine (CpG) -containing oligonucleotides and their physical cellular uptake is of mechanistic interest and a prerequisite for rational improvements of the therapeutic potential of CpG-harbouring oligonucleotides. Here, a combinatorial approach was used to identify nucleotide sequence motifs that facilitate increased cellular uptake in mammalian cells. Oligonucleotides harbouring the selected hexanucleotide TCGTGT in cis show increased cellular uptake. This motif contains a CpG dinucleotide within a sequence context that shows a very strong CpG-specific stimulatory activity on human B cells. Here we describe the influence of concentration, length and sequence position of the unmethylated CpG dinucleotide on immunostimulation. A comparison between phosphorothioate-derivatives and unmodified TCGTGT-containing oligonucleotides strongly indicates a great CpG-specificity for the unmodified CpG-harbouring oligonucleotides but not for the phosphorothioate versions. This work describes a link between the physical cellular uptake of naked oligonucleotides harbouring the selected cellular uptake motif TCGTGT, its strong CpG-specific stimulation of human B cells and its relationship with the sequence context of CpG and its cellular uptake.
Mende
Miriam
M
Institut für Molekulare Medizin, Universitätsklinikum Schleswig-Holstein and Universität zu Lübeck, Lübeck, Germany.
Hopert
Anne
A
Wünsche
Winfried
W
Overhoff
Marita
M
Detzer
Anke
A
Börngen
Kirsten
K
Schlenke
Peter
P
Kirchner
Holger
H
Sczakiel
Georg
G
D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism; D001483:Base Sequence; D002478:Cells, Cultured; D020650:Combinatorial Chemistry Techniques; D018899:CpG Islands / Q000235:genetics / Q000276:immunology; D004252:DNA Mutational Analysis; D004306:Dose-Response Relationship, Immunologic; D015723:Gene Library; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000276:immunology* / Q000378:metabolism; D008213:Lymphocyte Activation / Q000276:immunology; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000276:immunology* / Q000493:pharmacokinetics; D014407:Tumor Cells, Cultured
C408982:CPG-oligonucleotide; D009838:Oligodeoxyribonucleotides