Selection of oligonucleotide aptamers with enhanced uptake and activation of human leukemia B cells.
The clinical use of oligonucleotide (ODN) therapeutics has been hampered by their limited ability to penetrate intact cells. To identify ODN properties that would facilitate cellular uptake, we developed a repetitive selection procedure using an ODN library containing at least 10(14) different molecules and human B lymphoma cells as a target. Natural phosphodiester single-stranded DNA ODNs (R-aptamers) were obtained after 10 rounds of selection. A common feature in the R-aptamers was guanine-rich 3' terminal sequences, and many also contained potential immunostimulatory (ISS) CpG sequence motifs. Two R-aptamers (R10-60 and D-R15-8) with the predominant shared characteristics were selected for further study on primary human chronic lymphocytic leukemia (CLL) B cells, which are well known to be difficult to transfect and activate. Flow cytometry analysis of the CLL cells demonstrated that the fluorochrome-labeled R-aptamers were internalized much more efficiently than nonselected random sequence ODN. Studies on sequence modifications indicated that efficient uptake required ODN multimerization, that was promoted by guanine-rich sequences at the 3' terminus. In addition, CLL cells that were exposed to the aggregating R-aptamers containing CpG motifs were strongly activated, as indicated by upregulation of CD40 levels as compared to cells treated with nonaggregating CpG R-aptamers. Together, these findings suggest that the sequence compositions in R-aptamers that promote multimerization and contain optimal ISS CpG motifs facilitate the delivery of ISS-ODN to CLL cells and enhance the activation of these cells.
Wu
Christina C N
CC
Division of Rheumatology Allergy and Immunology, Department of Medicine, Sam and Rose Stein Institute for Research on Aging, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0663, USA. c5wu@ucsd.edu
Castro
Januario E
JE
Motta
Marina
M
Cottam
Howard B
HB
Kyburz
Diego
D
Kipps
Thomas J
TJ
Corr
Maripat
M
Carson
Dennis A
DA
D000276:Adjuvants, Immunologic / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D002499:Centrifugation, Density Gradient; D002851:Chromatography, High Pressure Liquid; D004268:DNA-Binding Proteins / Q000276:immunology / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D009841:Oligonucleotides / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D010539:Permeability; D011956:Receptors, Cell Surface / Q000032:analysis / Q000276:immunology / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051217:Toll-Like Receptor 9; D014407:Tumor Cells, Cultured
D000276:Adjuvants, Immunologic; C408982:CPG-oligonucleotide; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; D011956:Receptors, Cell Surface; C495371:TLR9 protein, human; C495370:Tlr9 protein, mouse; D051217:Toll-Like Receptor 9