Related Experiment Videos
P-chirality-dependent immune activation by phosphorothioate CpG oligodeoxynucleotides.
Arthur M Krieg1, Piotr Guga, Wojciech Stec
1Department of Internal Medicine, University of Iowa, and Veterans Affairs Medical Center, Iowa City, USA. akrieg@coleypharma.com
Oligonucleotides
|March 18, 2004
Summary
Chirality of phosphorothioate oligodeoxynucleotides (PS-oligos) impacts immune cell stimulation. Rp-chirality enhances early immune responses, while Sp-chirality provides longer-lasting effects due to improved stability.
Area of Science:
- Immunology
- Oligonucleotide Chemistry
- Molecular Biology
Background:
- Phosphorothioate oligodeoxynucleotides (PS-oligos) with CpG motifs stimulate immune cells via MAPK pathways.
- The chirality of phosphorus atoms in PS-oligos can influence their biological activities.
- Previous studies indicated sequence-context dependent immune stimulation by CpG PS-oligos.
Purpose of the Study:
- To investigate if the immune stimulation by CpG PS-oligos is dependent on the chirality of their internucleotide phosphorothioate linkages.
- To compare the effects of Rp and Sp stereoisomers on immune cell activation and signaling.
- To elucidate the role of P-chirality in CpG-mediated immune responses.
Main Methods:
- Synthesis of CpG PS-oligos with defined Rp or Sp chirality at phosphorothioate linkages.
- Assessment of mitogen-activated protein kinase (MAPK) activation and I kappa B degradation in immune cells.
- Measurement of cellular uptake rates of fluorescently labeled Rp and Sp CpG PS-oligos.
- Analysis of B cell proliferation and cytokine secretion using (3)H-thymidine incorporation assays over time.
Main Results:
- CpG PS-oligos with Rp configuration showed significantly stronger MAPK activation and I kappa B degradation at early time points (40 minutes) compared to Sp-linked oligos.
- Cellular uptake rates of Rp and Sp CpG PS-oligos were identical, ruling out differential uptake as the cause for enhanced stimulation.
- While Rp-chirality led to stronger initial stimulation, Sp-chirality was associated with sustained activity at 48 hours, suggesting improved stability.
- Stereodefined CpG dinucleotides were most stimulatory when Rp, indicating Rp-chirality at the CpG motif is crucial for early activation.
Conclusions:
- The sense of P-chirality in PS-oligos critically determines the biological activities of CpG motifs.
- Rp-chirality at the CpG dinucleotide is preferred for optimal early immune stimulation.
- Sp-chirality of the PS-oligo backbone appears to enhance stability, leading to more durable immune effects in prolonged assays.