Phosphodiester Oligodeoxynucleotides Research Articles

Expression of Concern: A Palindromic CpG-Containing Phosphodiester Oligodeoxynucleotide as a Mucosal Adjuvant Stimulates Plasmacytoid Dendritic Cell-Mediated TH1 Immunity.

Expression of Concern: A Palindromic CpG-Containing Phosphodiester Oligodeoxynucleotide as a Mucosal Adjuvant Stimulates Plasmacytoid Dendritic Cell-Mediated TH1 Immunity.

Effects of G-Quadruplex Ligands on the Topology, Stability, and Immunostimulatory Properties of G-Quadruplex-Based CpG Oligodeoxynucleotides.

We previously reported that the formation of guanine-quadruplex (G4) structures provides phosphodiester oligodeoxynucleotides containing unmethylated cytosine-phosphate-guanine (CpG ODNs) with higher nuclease resistance and cellular uptake, thereby increasing their immunostimulation efficiency through TLR9 activation. CpG ODNs forming G4 structures (G4 CpG ODNs) are thus potential vaccine adjuvants against infectious diseases. However, the G4 structure changes topology depending on the surrounding environment. Recently, G4 ligands, which are small molecules that bind to G4 ODNs with high affinity, were reported to improve the stability of G4. In this study, we propose to increase the stability and function of G4 CpG ODNs using G4 ligands. We show the effects of two G4 ligands, named L2H2-6OTD (L2H2) and L2G2-2M2EG-6OTD (L2G2), on the topology, stability, and immunostimulatory properties of a monomeric hybrid-type G4 CpG ODN containing CpG motifs in the central loop, named GD3. We found that L2H2 helps maintain the hybrid G4 topology of GD3, whereas L2G2 induces parallel G4 formation. Both G4 ligands increase the thermodynamic and nuclease stability of GD3. However, only GD3 associated with L2H2 binds efficiently to TLR9 and evokes a higher immune response from mouse macrophage-like RAW264 cells. GD3 associated with L2G2 does not bind efficiently to TLR9 and elicits lower cytokine production. Our results demonstrate that the potential to enhance immunostimulatory properties depends on the ability of G4 ligands to maintain and stabilize the hybrid G4 of GD3. We anticipate that our findings will facilitate the development of more effective G4 CpG ODN-based vaccine adjuvants against infectious diseases.

Elucidating the Motif for CpG Oligonucleotide Binding to the Dendritic Cell Receptor DEC-205 Leads to Improved Adjuvants for Liver-Resident Memory.

DEC-205 is a cell-surface receptor that transports bound ligands into the endocytic pathway for degradation or release within lysosomal endosomes. This receptor has been reported to bind a number of ligands, including keratin, and some classes of CpG oligodeoxynucleotides (ODN). In this study, we explore in detail the requirements for binding ODNs, revealing that DEC-205 efficiently binds single-stranded, phosphorothioated ODN of ≥14 bases, with preference for the DNA base thymidine, but with no requirement for a CpG motif. DEC-205 fails to bind double-stranded phosphodiester ODN, and thus does not bind the natural type of DNA found in mammals. The ODN binding preferences of DEC-205 result in strong binding of B class ODN, moderate binding to C class ODN, minimal binding to P class ODN, and no binding to A class ODN. Consistent with DEC-205 binding capacity, induction of serum IL-12p70 or activation of B cells by each class of ODN correlated with DEC-205 dependence in mice. Thus, the greater the DEC-205 binding capacity, the greater the dependence on DEC-205 for optimal responses. Finally, by covalently linking a B class ODN that efficiently binds DEC-205, to a P class ODN that shows poor binding, we improved DEC-205 binding and increased adjuvancy of the hybrid ODN. The hybrid ODN efficiently enhanced induction of effector CD8 T cells in a DEC-205-dependent manner. Furthermore, the hybrid ODN induced robust memory responses, and was particularly effective at promoting the development of liver tissue-resident memory T cells.

Synthetic Oligodeoxynucleotides in the Regulation of Neutrophil Apoptosis

Synthetic oligodeoxynucleotides (ODNs) mimic the immunostimulatory activity of viral/microbial/host DNA. The present study was undertaken to investigate the effects of ODNs on polymorphonuclear leukocyte (PMNL, neutrophil) apoptosis. Neutrophils are essential for innate immunity, and the ability of ODNs to influence neutrophil functions makes them promising for use in immunotherapy. Little is known about the effects of different ODN structures on neutrophil life and death. We studied how ODNs affect both unconstrained and bacterially suppressed apoptosis of neutrophilic granulocytes. Apoptosis modulation by phosphodiester ODNs and by structures with different numbers of phosphorothioate substitutions was compared. In the case of CpG-ODNs, it was the compounds with phosphorothioate bonds that had a pronounced proapoptotic effect upon short-term (4-6 hours) exposure. After 24 hours, the most effective were A-class CpG-ODNs, with phosphodiester inner core and phosphorothioate oligoguanosines (poly(g)) attached to the ends, as well as telomeric phosphodiester (TTAGGG)n repeats flanking with 3’- and 5’- poly(g) sequences, like those in A-class CpG-ODNs. Intracellular oxidant accumulation often triggers apoptotic pathways in neutrophils. Phosphorothioate-containing CpG-ODNs concomitantly stimulated production of nitric oxide and superoxide, which may rapidly combine to generate peroxynitrite. ODNs with phosphodiester bonds did not activate oxidant production. We experimentally showed the anti-apoptotic activity of bacteria Salmonella typhimurium. CpG-ODNs prevented bacteria-associated inhibition of apoptosis at doses that do not affect the lifespan of resting cells. The pro-oxidant effect of low concentrations of CpG ODNs was not sufficient to trigger irreversible pro-apoptotic mechanisms, but the sensitivity of PMNLs to ODNs as modulators of apoptosis, increased significantly under conditions of infectious inflammation. Telomeric phosphodiester (TTAGGG)4 repeats with phosphorothioate oligoguanosines attached to the ends were less effective in prevention of bacteria-associated inhibition of apoptosis than A-class CpG-ODNs with the same modified oligoguanosines overhangs. The results of our study revealed that synthetic analogs of bacterial/host DNA, when phosphorothioate modified, can be used as inducers of reactive oxygen species generation by neutrophils. This allows to overcome the excess pro-survival effect caused by some pathogens, in particular Salmonella typhimurium, thus facilitating the successful resolution of inflammation. Ethics statement. The authors prepared neutrophils from the blood of healthy volunteers. Blood was collected via venous puncture, as approved by the Ministry of Public Health Service of the Russian Federation. Experimental and the subject consent procedures were approved by the Ethics Committee of the A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University. Fully informed consent was obtained, and all investigations were conducted according to the principles of the Declaration of Helsinki.

Importance of probe design for bioanalysis of oligonucleotides using hybridization-based LC-fluorescence assays.

Aim: The importance of the length and/or structure of fluorescently labeled PNA (peptide nucleic acid) probes for quantitative determination of oligodeoxynucleotides (ODNs) is demonstrated in human plasma using hybridization-based LC-fluorescence assays. The length of the PNA probes impacts the peak shape and chromatographic separation of the resulting PNA/ODN hybridization complexes and affects assay sensitivity, dynamic range and carryover. Methods: For quantitative determination of an 18-mer phosphodiester ODN (DNL1818) in human plasma, an assay utilizing an Atto dye-labeled 12-mer PNA probe provided a linear quantitation range of 0.1-50ng/ml with excellent accuracy and precision (within -5.3-7.73%). Conclusion: This method provides a convenient method for sensitive and specific quantification of ODNs in biological matrix with limited sample volume and no special extraction.

Role of Cell-Penetrating Peptides in Intracellular Delivery of Peptide Nucleic Acids Targeting Hepadnaviral Replication.

Peptide nucleic acids (PNAs) are potentially attractive antisense agents against hepatitis B virus (HBV), although poor cellular uptake limits their therapeutic application. In the duck HBV (DHBV) model, we evaluated different cell-penetrating peptides (CPPs) for delivery to hepatocytes of a PNA-targeting hepadnaviral encapsidation signal (ε). This anti-ε PNA exhibited sequence-specific inhibition of DHBV RT in a cell-free system. Investigation of the best in vivo route of delivery of PNA conjugated to (D-Arg)8 (P1) showed that intraperitoneal injection to ducklings was ineffective, whereas intravenously (i.v.) injected fluorescein-P1-PNA reached the hepatocytes. Treatment of virus carriers with i.v.-administered P1-PNA resulted in a decrease in viral DNA compared to untreated controls. Surprisingly, a similar inhibition of viral replication was observed in vivo as well as in vitro in primary hepatocyte cultures for a control 2 nt mismatched PNA conjugated to P1. By contrast, the same PNA coupled to (D-Lys)4 (P2) inhibited DHBV replication in a sequence-specific manner. Interestingly, only P1, but not P2, displayed anti-DHBV activity in the absence of PNA cargo. Hence, we provide new evidence that CPP-PNA conjugates inhibit DHBV replication following low-dose administration. Importantly, our results demonstrate the key role of CPPs used as vehicles in antiviral specificity of CPP-PNA conjugates.

Effects of phosphodiester and phosphorothioate ODN2216 on leukotriene synthesis in human neutrophils and neutrophil apoptosis.

Polymorphonuclear leukocytes (PMNLs, neutrophils) play a major role in the initiation and resolution of the inflammatory response, and neutrophil apoptosis is a critical step in resolving inflammation. We examined the effects of oligodeoxynucleotide (ODN) species with different numbers of phosphodiester and phosphorothioate bonds on leukotriene synthesis in PMNLs and on neutrophil apoptosis. Our modifications were based on the well-known ODN2216 molecule (Krug etal., 2001). Treatment of cultured human neutrophils with ODN2216 accelerated apoptosis except in the case of a species with only phosphodiester bonds. The ODNs with poly(g) (phosphorothioate) sequences at both ends and a phosphodiester inner core had maximal effects on leukotriene synthesis in neutrophils and inhibited formation of 5-lipoxygenase metabolites. Addition of phosphodiester and phosphorothioate ODNs to PMNLs produced distinct effects on superoxide and nitric oxide formation: phosphorothioate-containing ODNs concomitantly stimulated production of nitric oxide and superoxide, which may rapidly combine to generate peroxynitrite. Altogether, our results describe strong activation of neutrophil's cellular responses by phosphorothioate ODN2216. We propose that phosphorothioate modification of ODNs represents a potential mechanism of PMNL activation.

A palindromic CpG-containing phosphodiester oligodeoxynucleotide as a mucosal adjuvant stimulates plasmacytoid dendritic cell-mediated T(H)1 immunity.

BackgroundCpG oligodeoxynucleotides (ODNs), resembling bacterial DNA, are currently tested in clinical trials as vaccine adjuvants. They have the nuclease-resistant phosphorothioate bond; the immune responses elicited differ according to the CpG ODN sequence and vaccination method. To develop a CpG ODN that can induce plasmacytoid dendritic cell (pDC)-mediated TH1 immunity through the mucosa, we constructed phosphodiester G9.1 comprising one palindromic CpG motif with unique polyguanosine-runs that allows degradation similar to naturally occurring bacterial DNA.MethodsTH1 and TH2 immunity activation was evaluated by cytokine production pattern and T-bet/GATA-3 ratio in human peripheral blood mononuclear cells and mouse bone marrow cells. Adjuvanticity was evaluated in mice administered G9.1 with diphtheria toxoid (DT) through nasal vaccination.ResultsG9.1 exhibited stronger IFN-α-inducing activity than A-class CpG ODN2216 and increased T-bet/GATA-3 ratio by enhancing T-bet expression. Nasally administered G9.1 plus DT induced DT-specific mucosal IgA and serum IgG, but not IgE, responses with antitoxin activity in C57BL/6 and BALB/c mice, possibly due to IFN/BAFF production. Induction of TH1, but not TH2, -type Abs depended completely on pDCs, the first in vivo demonstration by CpG ODNs.ConclusionsG9.1 is a promising mucosal adjuvant for induction of pDC-mediated TH1 immunity.

Abstract 1046: AS1411 activity is regulated by epidermal growth factor receptor signaling pathway.

Abstract The anticancer AS1411 agent is a G-rich phosphodiester oligodeoxynucleotide, which forms a stable quadruplex structure and binds specifically to nucleolin as an aptamer. It efficiently inhibits proliferation and induces cell death in many types of cancer cells, but has little effect on normal cells. We have also shown that AS1411 is taken up by macropinocytosis and stimulates further macropinocytosis by a nucleolin-dependent mechanism in several cancer cells. AS1411 activity correlates with stimulated macropinocytosis, suggesting this hyperstimulation of macropinocytosis may explain the unusual cancer cell death caused by AS1411. Macropinocytosis is a ligand-independent endocytic pathway that is normally activated by growth factor receptor stimulation. On the other hand, nucleolin has been reported that can enhance growth factor signaling by binding directly to some growth factor receptors. Therefore in this study, we determine the participation of growth factor signaling pathway in AS1411-induced macropinocytosis and cell death. Pre-incubation of DU145 cells with a specific EGFR inhibitor, but not PDGFR or JAK2 inhibitors, significantly inhibited the stimulation of AS1411-mediated macropinocytosis, suggesting that AS1411 activates EGFR signaling pathways. This was confirmed by observing that DU145 cells treated with AS1411 induced tyrosine phosphorylation of EGFR and activation of its downstream effectors, including p38, PI3K and Rac1. Furthermore, AS1411 seem to induce the interaction between EGFR and nucleolin in accord with EGFR activation. We also found that the activation of EGFR signaling pathway by AS1411 might regulate its cancer cellular response. Simulation of macropinocytosis by AS1411 was significantly inhibited in DU145 cells pre-treated with pharmacological inhibitors of Rac1 or PI3K, but not by Src inhibitor. Moreover the ability of AS1411 to inhibit cell proliferation and induce cell death was reduced by an EGFR-neutralizing antibody (cetuximab) or by downregulating the expression of Rac1 using siRNAs. These results indicate that AS1411 mediates the activation of EGFR signaling pathway to induce cancer cell death, and suggest that AS1411 might redirect EGFR signaling pathway from a cell survival signal to a cell death signal. Citation Format: Elsa M. Reyes-Reyes, Francesca R. Salipur, Paula J. Bates. AS1411 activity is regulated by epidermal growth factor receptor signaling pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1046. doi:10.1158/1538-7445.AM2013-1046

Nuclease-resistant immunostimulatory phosphodiester CpG oligodeoxynucleotides as human Toll-like receptor 9 agonists

BackgroundUnmethylated cytosine-guanine (CpG) motif-containing oligodeoxynucleotides (ODNs) have been well characterized as agonists of Toll-like receptor 9 (TLR9). ODNs with a phosphorothioate (PTO) backbone have been studied as TLR9 agonists since natural ODNs with a phosphodiester (PD) backbone are easily degraded by a serum nuclease, which makes them problematic for therapeutic applications. However, ODNs with a PTO backbone have been shown to have undesirable side effects. Thus, our goal was to develop nuclease-resistant, PD ODNs that are effective as human TLR9 (hTLR9) agonists.ResultsThe sequence of ODN2006, a CpG ODN that acts as an hTLR9 agonist, was used as the basic CpG ODN material. The 3'-end modification of ODN2006 with a PD backbone (PD-ODN2006) improved its potential as an hTLR9 agonist because of increased resistance to nucleolytic degradation. Moreover, 3'-end modification with oligonucleotides showed higher induction than modification with biotin, FITC, and amino groups. Further, enhancement of hTLR9 activity was found to be dependent on the number of CpG core motifs (GTCGTT) in the PD ODN containing the 3'-end oligonucleotides. In particular, ODN sequences consisting of two to three linked ODN2006 sequences with a PD backbone (e.g., PD-ODN2006-2006 and PD-ODN2006-2006-2006) acted as effective agonists of hTLR9 even at lower concentrations.ConclusionsThis study showed that PD-ODN2006-2006 and PD-ODN-2006-2006-2006 can be used as potentially safe agonists for hTLR9 activation instead of CpG ODNs with a PTO backbone. We propose these CpG ODNs consisting of only a PD backbone as a novel class of CpG ODN.

Novel immunostimulatory phosphodiester oligodeoxynucleotides with CpT sequences instead of CpG motifs

The innate immune system recognizes bacterial DNA as a nonself to induce rapid immune activation. TLR9 recognizes synthetic oligodeoxynucleotides (ODNs) and bacterial DNA containing unmethylated CpG dinucleotides in the context of specific base sequences (CpG-DNA). Here, we demonstrate that phosphorothioate backbone CT-ODN (PS-CT-ODN), a derivative of phosphorothioate backbone CpG-DNA (PS-ODN) with CT sequences substituted for the CG sequences, stimulates IL-8 promoter activation and gene expression. Furthermore, we identified an immunostimulatory phosphodiester bond CT-ODN (PO-CT-ODN) from Staphylococcus aureus chromosomal DNA and found that the PO-CT-ODN induces cytokine production in a TLR9-dependent manner when encapsulated with a proper liposome. Our experimental analyses also demonstrate that the immunostimulatory PO-CT-ODN can act as an adjuvant for the induction of Ag-driven IgG production. Further investigation of the functional role of PO-CT-ODN may support the future application of PO-CT-ODN in immunotherapeutics.

The immunostimulatory activity of phosphorothioate CpG oligonucleotides is affected by distal sequence changes

CpG motifs in bacterial DNA activate innate immune cells via toll like receptor 9 (TLR9). Short synthetic oligodeoxynucleotides (ODN) containing a six base CpG motif can mimic the immunostimulatory activity of bacterial DNA. Phosphorothioate (PS) modification of the backbone of ODN makes them more resistant to nuclease degradation and consequently preferable for therapeutic use. Previous studies have shown that the sequence requirements for PS-ODN to have maximal stimulatory activity are more stringent than for normal phosphodiester (PO) ODN. Here we show small sequence changes distal to the CpG motif can affect the activity of PS-ODN whilst having no effect on the activity of PO-ODN. The addition of terminal dG residues and other minor changes to the potently immunostimulatory PS-ODN 1668S caused delayed signalling. The reduction in immunostimulatory activity of PS-ODN was associated with a delay in the activation of MAP kinases.

Structural and immunostimulatory properties of Y-shaped DNA consisting of phosphodiester and phosphorothioate oligodeoxynucleotides

Y-shape formation increased the immunostimulatory activity of phosphodiester (PO) oligodeoxynucleotides (ODNs) containing CpG motif. In this study, PO CpG ODN or CpG ODN containing nuclease-resistant phosphorothioate (PS) linkages, i.e., PS CpG ODN or PO CpG ODN with three PS linkages at the both ends (PS3), was mixed with two PO- or PS ODNs to prepare Y-shaped DNA (Y-DNA) containing a potent CpG motif. The melting temperature of Y-DNA decreased with increasing number of PS linkages. Y(PS/PO/PO), which contained PS CpG ODN, showed the greatest activity to induce tumor necrosis factor-α release from macrophage-like RAW264.7 cells, followed by Y(PS3/PO/PO). However, the high activity of Y(PS/PO/PO) was due to that of PS CpG ODN, and Y-shape formation had no significant effect on the activity. Furthermore, PS CpG ODN of Y(PS/PO/PO) was efficiently taken up by cells, but other PO ODNs in the Y-DNA were not, indicating that PS CpG ODN in Y-DNA behave like single stranded PS CpG ODN. In quite contrast, the immunostimulatory activity of PS3 CpG ODN was significantly increased by Y-shape formation. In conclusion, Y-shape formation and PS substitution can be used simultaneously to increase the immunostimulatory activity of CpG ODN, but extensive substitution should be avoided because it diminishes the benefits of Y-shape formation.

Abstract 4450: A new paradigm for AS1411 activity: Uptake by macropinocytosis and induction of macropinocytosis by a nucleolin-dependent mechanism

Abstract AS1411 is an experimental drug that is currently in Phase II clinical trials as a novel therapy for cancer. It is a G-rich phosphodiester oligodeoxynucleotide, which forms a stable quadruplex structure and binds specifically to nucleolin as an aptamer. We have shown previously that AS1411 can efficiently inhibit proliferation and induce cell death in many types of cancer cells, but has little effect on normal cells. Here, we report on the uptake of fluorescently labeled AS1411 (FL-AS1411) in a variety of cancer cell lines (sensitive to AS1411) and non-malignant cells (resistant to AS1411), which we studied using flow cytometry and confocal microscopy. In both cancer and non-malignant cells, uptake of FL-AS1411 was much higher that that of an inactive control oligonucleotide and was rapid (clearly visible in cytoplasmic foci by 15 min). Uptake of FL-AS1411 (evaluated at 2 h) was independent of the presence of serum, but strongly temperature-dependent, indicating an active process. In cancer cells, FL-AS1411 uptake was significantly suppressed by macropinocytosis inhibitor, amiloride, but not by dynamin inhibitor, dynasore, and confocal microscopy showed that AS1411 co-localized mainly with the macropinocytic tracker, dextran, but not with transferrin (a marker of receptor-mediated endocytosis). In contrast, in non-malignant cells, FL-AS1411 uptake was significantly suppressed by dynasore, but not by amiloride. These results suggest that the major mechanism of internalization of AS1411 occurs via macropinocytosis in cancer cells, but via clathrin-dependent and/or caveolae-dependent pathways in normal cells. Additionally, we found that treatment of cancer cells with an oligonucleotide containing the AS1411 sequence caused hyperstimulation of macropinocytosis, provoking an increment in its own uptake. No stimulation of macropinocytosis was observed in non-malignant cells, which are insensitive to AS1411. By using siRNA approaches, we showed that nucleolin is not required for the initial phase of FL-AS1411 uptake (at 2 h), but it is necessary for the induced macropinocytosis and consequently for the second phase of FL-AS1411 uptake (at 24 h). These data confirm the important role of nucleolin in mediating the activity of AS1411 and provide a new model for the mechanism of action of AS1411 in cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4450.

LL-37 Promotes Rapid Sensing of CpG Oligodeoxynucleotides by B Lymphocytes and Plasmacytoid Dendritic Cells

LL-37 is a cationic antimicrobial peptide derived from neutrophils and keratinocytes. It plays an important role in protection against bacterial infection in the skin and mucosal surfaces. However, its role within the blood compartment remains unclear given that serum inhibits its bactericidal property. In this study, we show that LL-37 promotes very rapid and highly efficient sensing of CpG motifs in bacterial DNA by human B lymphocytes and plasmacytoid dendritic cells (pDCs) in serum-containing media and in whole blood. LL-37 allowed detection of CpG oligodeoxynucleotide (ODN) within minutes of exposure. Without LL-37, 20-30 times more CpG was required to produce the same effect. The promotion of CpG detection by LL-37 was independent of the backbone of the ODN, as the effect was observed not only in ODNs with modified phosphorothioate backbone, but also in ODNs with natural phosphodiester backbone, as found in genomic DNA. Unmethylated CpG motifs within the phosphodiester ODN and LL-37-mediated delivery are required for pDCs to respond. In keeping with the above, cells responded to CpG-rich bacterial DNA and LL-37, but not to human DNA and LL-37. The ability of LL-37 to enhance delivery of CpG to stimulate immune cells is independent of its amphipathic structure and its bactericidal property. LL-37 aids the delivery of CpG to B cells and pDCs, but not T cells. These findings are pertinent to rapid recognition of microbial DNA and are highly relevant to contemporary studies of CpG/TLR9 agonists in vaccines and cancer therapy.

Higher activation of TLR9 in plasmacytoid dendritic cells by microbial DNA compared with self-DNA based on CpG-specific recognition of phosphodiester DNA

TLR9 detects DNA in endolysosomal compartments of human B cells and PDC. Recently, the concept of the CpG motif specificity of TLR9-mediated detection, specifically of natural phosphodiester DNA, has been challenged. Unlike in human B cells, CpG specificity of natural phosphodiester DNA recognition in human PDC has not been analyzed in the literature. Here, we found that the induction of IFN-alpha and TNF-alpha in human PDC by phosphodiester ODNs containing one or two CG dinucleotides was reduced to a lower level when the CG dinucleotides were methylated and was abolished if the CGs were switched to GCs. Consistent with a high frequency of unmethylated CG dinucleotides, bacterial DNA induced high levels of IFN-alpha in PDC; IFN-alpha was reduced but not abolished upon methylation of bacterial DNA. Mammalian DNA containing low numbers of CG dinucleotides, which are frequently methylated, induced IFN-alpha in PDC consistently but on a much lower level than bacterial DNA. For activation of PDC, phosphodiester ODNs and genomic DNA strictly required complexation with cationic molecules such as the keratinocyte-derived antimicrobial peptide LL37 or a scrambled derivative. In conclusion, we demonstrate that self-DNA complexed to cationic molecules activate PDC and thus, indeed, may function as DAMPs; nevertheless, the preference of PDC for CpG containing DNA provides the basis for the discrimination of microbial from self-DNA even if DNA is presented in the condensed form of a complex.

Sequence independent interferon‐α induction by multimerized phosphodiester DNA depends on spatial regulation of Toll‐like receptor‐9 activation in plasmacytoid dendritic cells

Single-stranded versus multimeric phosphorothioate-modified CpG oligodeoxynucleotides (ODNs) undergo differential endosomal trafficking upon uptake into plasmacytoid dendritic cells (pDCs), correlating with Toll-like receptor-9-dependent pDC maturation/activation (single-stranded B-type CpG ODN) or interferon-alpha (IFN-alpha) induction (multimeric A-type CpG ODN), respectively. As was recently shown, IFN-alpha production, other than by CpG ODNs, can also be induced in a sequence-independent manner by phosphodiester (PD) ODNs multimerized by 3' poly-guanosine (poly-G) tails. We investigate here the type of endosomal trafficking responsible for IFN-alpha induction by natural PD ODN ligands. We show that 3' extension with poly-G tails leads to multimerization of single-stranded PD ODNs and to enhanced cellular uptake into pDCs. While monomeric PD ODNs, which induce CpG-dependent Toll-like receptor-9 stimulation and pDC maturation/activation, localized to late endosomal/lysosomal compartments, the poly-G multimerized PD ODNs, which induce CpG-independent IFN-alpha production, located to vesicles with a distinct, 'early' endosomal phenotype. We conclude that poly-G-mediated multimerization of natural PD ODNs allows for sequence-independent, Toll-like receptor-9-dependent IFN-alpha induction in pDCs by combining three distinct effects: relative protection of sensitive PD ODNs from extracellular and intracellular DNase degradation, enhanced cellular uptake and preferential early endosomal compartmentation.

Effect of Dendritic Cells Treated with CpG ODN on Atopic Dermatitis of Nc/Nga mice

Atopic dermatitis (AD) is a chronic inflammatory skin disease and the pathogenesis of AD is associated with the release of various cytokines/chemokines due to activated Th(2) immune responses. Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotide in the context of particular base sequence (CpG motifs) are known to have the immunostimulatory activities in mice and to convert from Th(2) to Th(1) immune responses in AD. We aimed to investigate that CpG ODN, especially phosphodiester form, can stimulate the protective immunity in NC/Nga mice with AD. We isolated BMDCs from NC/Nga mice and then, cultured with GM-CSF and IL-4 for 6 days, and treated for 2 days by either phosphorothioate ODN or phosphodiester ODN. CpG ODN-treated DCs resulted in more production of IL-12. When CpG ODN-treated DCs were intravenously injected into the NC/Nga mice, the NC/Nga mice with CpG ODN-treated DCs showed significant improvement of AD symptoms and decrease of IgE level. Histopathologically, the NC/Nga mice skin with CpG ODN-treated DCs showed the decreased IL-4 and TARC expression comparing with non-injected mice. These results may suggest that phosphodiester CpG ODN-treated DCs might function as a potent adjuvant for AD in a mouse model.

Nucleotide Pyrophosphatase/Phosphodiesterase 1 Is Responsible for Degradation of Antisense Phosphorothioate Oligonucleotides

The rapid degradation of unmodified phosphodiester oligodeoxynucleotides (PO-oligos) by exo -and endonucleases limits their application as antisense constructs and requires the synthesis and use of modified oligonucleotides. Phosphorothioate analogs of oligonucleotides (PS-oligos) are much more stable against nucleolytic degradation than their unmodified counterparts, and this is one of the reasons for which they are a promising class of antisense oligonucleotides. However, PS-oligos also undergo slow hydrolysis by enzymes present in plasma. The oligonucleotide degradation proceeds mainly from the 3' -end, resulting in the formation of a typical ladder of shorter products and the release of the mononucleoside 5' -phosphorothioates. So far, little has been known concerning the molecular identity of the enzymes involved in the degradation of PS-oligos. We now identify the human plasma 3' -exonuclease responsible for their degradation as a soluble form of nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) (EC 3.1.4.1/EC 3.6.1.9), also known as the plasma cell differentiation antigen PC-1. We also show that adenosine or deoxyadenosine (alpha-thio)triphosphates can act as potent inhibitors of NPPs.

Separation of Phosphodiester Oligodeoxynucleotides and Phosphorothioate Antisense Oligodeoxynucleotides by Capillary Zone Electrophoresis at a Low pH

Oligodeoxynucleotides (ODNs) may possess biological activity in vivo, and are used for the cancer therapeusis. Synthesized ODNs contains many by-products, and so their purity check and resolution of single-base, i. e., the separation of ODNs differing by one nucleotide in length, become necessary. In this study, capillary zone electrophoresis (CZE) method was developed for the separation of two sets of model compounds of single-stranded oligodeoxynucleotide mixtures (18 - 20 mers), phosphodiester oligodeoxynucleotides (PO-ODNs) and their phosphorothioate modifications (PS-ODNs), with equal sequences differing in a single base. The effects of the CZE operating parameters on the separation were investigated and optimized to further improve the resolution, such as the pH values and the concentrations of running buffer, the varieties and concentrations of additives, the separation voltage as well as the temperature. It was confirmed that the pH value of the buffer played the most important role in the separation, and the urea used as the additive in the system improved significantly the resolution of PS-ODNs. Consequently, the PO-ODNs and PS-ODNs mixtures could be single-based separated on a fused-silica capillary of 50 microm x 49.0 cm (40.7 cm of effective length) under the optimum conditions: the running buffer system of 50 mmol/L NaH2PO4-H3PO4 (pH 2.24)-7 mol/L urea, the pressure injection of 2 kPa x 10 s, the separation voltage of -20 kV, the column temperature of 25 degrees C, and the ultraviolet (UV) detection at 260 nm. The average resolutions for the separation of 18 - 19 mers and 19 - 20 mers of PO-ODNs were 4.68 and 3.20, respectively; and the average resolutions for the separation of 18 - 19 mers and 19 - 20 mers of PS-ODNs were 1.23 and 0.81, respectively. The relative standard deviations of the migration time and the resolution were all less than 5%. This method will be useful for the qualification of PO-ODNs and PS-ODNs samples as they are used in antisense drug development due to the relatively easy operation and good reproducibility of the method in comparing with the capillary gel electrophoresis.