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Hairpin oligodeoxynucleotides incorporating a (2R,3S)-4-(methoxyamino)butane-1,2,3-triol residue in the middle of the double-helical stem and opposite to either one of the canonical nucleobases or an abasic 2-(hydroxymethyl)tetrahydrofuran-3-ol spacer were synthesized. Under mildly acidic conditions, aromatic aldehydes reacted reversibly with these oligonucleotides, converting the (2R,3S)-4-(methoxyamino)butane-1,2,3-triol unit into a 2-aryl-N-methoxy-1,3-oxazinane nucleoside analogue. The equilibrium of this reaction was found to be dependent on both the aldehyde and the nucleobase opposite to the modified residue. 9-Formyl-9-deazaadenine, combining a large stacking surface with an array of hydrogen bond donors and acceptors, showed the highest affinity as well as selectivity consistent with the rules of Watson-Crick base pairing. 5-Formyluracil or indole-3-carbaldehyde, lacking in either stacking or hydrogen bonding ability, were incorporated with a much lower affinity and selectivity., (© 2023The Authors. Published by Wiley-VCH GmbH.)

Specific chemical reactions by enzymes acting on a nucleobase are realized by flipping the target base out of the helix. Similarly, artificial oligodeoxynucleotides (ODNs) can also induce the base flipping and a specific chemical reaction. We now report an easily prepared and unique structure-providing photo-cross-linking reaction by taking advantage of the base-flipping-out field formed by alkene-type base-flipping-inducing artificial bases. Two 3-arylethenyl-5-methyl-2-pyridone nucleosides with the Ph or An group were synthesized and incorporated into the ODNs. We found that the two Ph derivatives provided the cross-linked product in a high yield only by a 10 s photoirradiation when their alkenes overlap each other in the duplex DNA. The highly efficient reaction enabled forming a cross-linked product even when using the duplex with a low T m value.

MYC , originally named c- myc , is an oncogene deregulated in many different forms of cancer. Translocation of the MYC gene to an immunoglobulin gene leads to an overexpression and the development of Burkitt's lymphoma (BL). Sporadic BL constitutes one subgroup where one of the translocation sites is located at the 5'-vicinity of the two major MYC promoters P₁ and P₂. A non-B-DNA forming sequence within this region has been reported with the ability to form an intramolecular triplex (H-DNA) or a G-quadruplex. We have examined triplex formation at this site first by using a 17 bp triplex-forming oligonucleotide (TFO) and a double strand DNA (dsDNA) target corresponding to the MYC sequence. An antiparallel purine-motif triplex was detected using electrophoretic mobility shift assay. Furthermore, we probed for H-DNA formation using the BQQ-OP based triplex-specific cleavage assay, which indicated the formation of the structure in the supercoiled plasmid containing the corresponding region of the MYC promoter. Targeting non-B-DNA structures has therapeutic potential; therefore, we investigated their influence on strand-invasion of anti-gene oligonucleotides (ON)s. We show that in vitro, non-B-DNA formation at the vicinity of the ON target site facilitates dsDNA strand-invasion of the anti-gene ONs.

Human alkyladenine DNA glycosylase (AAG) protects DNA from alkylated and deaminated purine lesions. AAG flips out the damaged nucleotide from the double helix of DNA and catalyzes the hydrolysis of the N-glycosidic bond to release the damaged base. To understand better, how the step of nucleotide eversion influences the overall catalytic process, we performed a pre-steady-state kinetic analysis of AAG interaction with specific DNA-substrates, 13-base pair duplexes containing in the 7th position 1-N6-ethenoadenine (εA), hypoxanthine (Hx), and the stable product analogue tetrahydrofuran (F). The combination of the fluorescence of tryptophan, 2-aminopurine, and 1-N6-ethenoadenine was used to record conformational changes of the enzyme and DNA during the processes of DNA lesion recognition, damaged base eversion, excision of the N-glycosidic bond, and product release. The thermal stability of the duplexes characterized by the temperature of melting, T m , and the rates of spontaneous opening of individual nucleotide base pairs were determined by NMR spectroscopy. The data show that the relative thermal stability of duplexes containing a particular base pair in position 7, (T m (F/T) < T m (εA/T) < T m (Hx/T) < T m (A/T)) correlates with the rate of reversible spontaneous opening of the base pair. However, in contrast to that, the catalytic lesion excision rate is two orders of magnitude higher for Hx-containing substrates than for substrates containing εA, proving that catalytic activity is not correlated with the stability of the damaged base pair. Our study reveals that the formation of the catalytically competent enzyme-substrate complex is not the bottleneck controlling the catalytic activity of AAG.

Unmethylated CpG oligodeoxynucleotides induce inflammatory immune responses through cytokine production and have attracted increasing attention as an immunostimulator. However, there remains a challenging issue of the use of 'native CpG DNA'. In the present study, we prepared cationic nanometer-sized gels (nanogels) consisting of cycloamylose modified with cholesterol and diethylaminoethane to form hydrophobic cross-linking points and to add positively charged groups, respectively. The cationic nanogels and native CpG DNA formed nanometer-sized complexes. Complexes of native CpG DNA with cationic nanogels delivered native CpG DNA to macrophage-like cells and induced cytokine production. In addition, complexes of negative control oligonucleotides with cationic nanogels did not induce cytokine production, and the induction of cytokines using complexes of phosphorothioate-modified CpG with cationic nanogels was lower than that of native CpG DNA. These results suggest that the complex of native CpG DNA with cationic nanogels is a promising strategy for nucleic acid adjuvants.

A study on binding of antitumor chelerythrine to human telomeric DNA/RNA G-quadruplexes was performed by using DNA polymerase stop assay, UV-melting, ESI-TOF-MS, UV-Vis absorption spectrophotometry and fluorescent triazole orange displacement assay. Chelerythrine selectively binds to and stabilizes the K(+)-form hybrid-type human telomeric DNA G-quadruplex of biological significance, compared with the Na(+)-form antiparallel-type DNA G-quadruplex. ESI-TOF-MS study showed that chelerythrine possesses a binding strength for DNA G-quadruplex comparable to that of TMPyP4 tetrachloride. Both 1:1 and 2:1 stoichiometries were observed for chelerythrine's binding with DNA and RNA G-quadruplexes. The binding strength of chelerythrine with RNA G-quadruplex is stronger than that with DNA G-quadruplex. Fluorescent triazole orange displacement assay revealed that chelerythrine interacts with human telomeric RNA/DNA G-quadruplexes by the mode of end- stacking. The relative binding strength of chelerythrine for human telomeric RNA and DNA G-quadruplexes obtained from ESI-TOF-MS experiments are respectively 6.0- and 2.5-fold tighter than that with human telomeric double-stranded hairpin DNA. The binding selectivity of chelerythrine for the biologically significant K(+)-form human telomeric DNA G-quadruplex over the Na(+)-form analogue, and binding specificity for human telomeric RNA G-quadruplex established it as a promising candidate in the structure-based design and development of G-quadruplex specific ligands.

Detection of DNA damage has been greatly improved following the development of equipment and techniques, however, discrimination between 5-hydroxymethylcytosine (5-hmC) and 5-methylcytosine (5-mC) is still a thorny problem. In the present study, an approach to oxidize and selective label (Ox-Labeling) 5-hmC in native DNA has been reported, which conveniently distinguishes 5-hmC from 5-mC using simple and effective processes., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

This unit describes procedures for the synthesis of a dinucleotide-type building block of the pyrimidine(6-4)pyrimidone photoproduct [(6-4) photoproduct], which is one of the major DNA lesions induced by ultraviolet (UV) light, and its incorporation into oligodeoxyribonucleotides. Although this type of lesion is frequently found at thymine-cytosine sites, the building block of the (6-4) photoproduct formed at thymine-thymine sites can be synthesized much more easily. The problem in the oligonucleotide synthesis is that the (6-4) photoproduct is labile under alkaline conditions. Therefore, building blocks with an amino-protecting group that can be removed by a brief treatment with ammonia water at room temperature must be used for the incorporation of the normal bases. Byproduct formation by the coupling of phosphoramidites with the N3 of the 5' component should also be considered. This side reaction can be avoided by using benzimidazolium triflate as an activator.

DNA-based immunomodulatory sequences (DIMS) are promising compounds for the treatment of different diseases, including inflammation and cancer. They act through the interaction with TLR9, a member of the Toll-like receptor family whose essential role in innate immunity was recently recognised by being awarded the Nobel Prize 2011. Combining the data obtained from in vitro and in vivo models with circular dichroism spectroscopy approach, we could show that formation of certain tertiary structures by DIMS can be connected to their specific physiologic effects such as activation of immune cells, induction of interferons and delay of the disease progression. Moreover the ability of selected DIMS compounds to form certain tertiary structures must be regarded as important for biological activities as is the presence of functional primary structure motifs such as unmethylated deoxyribodinucleotide CpG. These findings are useful when considering the design of DNA-based immunomodulators.

Nucleophosmin (NPM1) is a nucleocytoplasmic shuttling protein, mainly localized at nucleoli, that plays a key role in several cellular functions, including ribosome maturation and export, centrosome duplication, and response to stress stimuli. More than 50 mutations at the terminal exon of the NPM1 gene have been identified so far in acute myeloid leukemia; the mutated proteins are aberrantly and stably localized in the cytoplasm due to high destabilization of the NPM1 C-terminal domain and the appearance of a new nuclear export signal. We have shown previously that the 70-residue NPM1 C-terminal domain (NPM1-C70) is able to bind with high affinity a specific region at the c-MYC gene promoter characterized by parallel G-quadruplex structure. Here we present the solution structure of the NPM1-C70 domain and NMR analysis of its interaction with a c-MYC-derived G-quadruplex. These data were used to calculate an experimentally restrained molecular docking model for the complex. The NPM1-C70 terminal three-helix bundle binds the G-quadruplex DNA at the interface between helices H1 and H2 through electrostatic interactions with the G-quadruplex phosphate backbone. Furthermore, we show that the 17-residue lysine-rich sequence at the N terminus of the three-helix bundle is disordered and, although necessary, does not participate directly in the contact surface in the complex.

Objective: An immunomodulatory activity of circulating DNA (cirDNA) is implemented via the interactions of cirDNA with the targets exposed on the cell membrane and/or intracellular targets. The goal of this work was to identify the cellular targets of immunoinhibiting cell-surface-bound cirDNA (csbDNA) using its oligodeoxyribonucleotide (ODN) analogs containing the nucleotide motifs frequently found in csbDNA and displaying the same effects., Materials and Methods: The binding of [(32)P]-labeled single- and double-stranded ODNs (ss- and ds-ODNs) with membrane-cytosolic (MC) extracts and living human umbilical vein endothelial cells (HUVEC) was studied by electromobility shift assay (EMSA). Complexes of biotinylated ODNs with target proteins were affinity isolated using streptavidin Sepharose with subsequent SDS-PAGE and identified by MALDI-TOF mass spectrometry., Results and Conclusions: Both ss- and ds-ODNs form strong ODN-protein complexes with similar electrophoretic mobilities after incubation with the MC extracts of HUVEC either when added extracellularly or lipofected into cells. The ODN-binding proteins were identified as the DNA-binding components of DNA-dependent protein kinase (DNA-PK), namely, Ku70 and Ku80 proteins. Diverse cellular localizations and functions of the Ku proteins demand further clarification of Ku70/80 role as a mediator of the csbDNA immunoinhibiting effects.

Atopic dermatitis (AD) is a common, chronically relapsing skin disease. It is very difficult to treat severe type AD by steroid ointment. We therefore hypothesized that synthetic double-stranded DNA with a high affinity for Signal Transducers and Activators of Transcription 6 (STAT6) could be introduced in vivo as a decoy cis elements to bind the transcriptional factor and to block the gene activation of contributing the onset and progression of AD, thus providing effective therapy for AD. Treatment by the transfection of STAT6 decoy oligodeoxynucleotides (ODN), but not scrambled decoy ODN in the AD model mice, had a significantly inhibitory effect on not only STAT6 binding to nuclei but also on the third phase response. A histological analysis revealed that both edema and the infiltration of eosinophils and degranulated mast cells significantly decreased in STAT6 decoy ODN transfected mice. We herein report the first successful in vivo transfer of STAT6 decoy ODN to reduce the third phase reaction in AD model mice, thereby providing a new therapeutic strategy for atopic dermatitis. We also introduce another NF-kB Decoy Oligodeoxynucleotides therapy or STAT6siRNA therapy for allergic diseases.

Purpose: The Toll-like receptor (TLR) 9 ligand CpG has been used successfully for the immunotherapy of cancer. Chronic CpG application in tumor-free hosts leads, however, to the expansion of myeloid-derived suppressor cells (MDSC), which can cause T-cell suppression and may thus hamper the development of an effective immune response. Here, we investigated the effect of TLR9 activation on the function of MDSC in tumor-bearing mice., Experimental Design: We investigated the effect of CpG treatment on the number, phenotype, and function of MDSC in mice bearing subcutaneous C26 tumors and in CEA424-TAg mice bearing autochthonous gastric tumors., Results: CpG treatment blocks the suppressive activity of MDSC on T-cell proliferation in both tumor models. Inhibition of MDSC function by CpG was particularly pronounced for a highly suppressive Ly6G(hi) polymorphonuclear subset of MDSC. We further show that TLR9 activation by CpG promotes maturation and differentiation of MDSC and strongly decreases the proportion of Ly6G(hi) MDSC in both tumor-bearing and tumor-free mice. We demonstrate that IFN-α produced by plasmacytoid dendritic cells upon CpG stimulation is a key effector for the induction of MDSC maturation in vitro and show that treatment of mice with recombinant IFN-α is sufficient to block MDSC suppressivity., Conclusions: We show here for the first time that TLR9 activation inhibits the regulatory function of MDSC in tumor-bearing mice and define a role for the antitumoral cytokine IFN-α in this process.

The absence or insufficiency of specific immune response results in chronic hepatitis B virus (HBV) infection and immunotolerance. Therapeutic fusion peptide containing hepatitis B core antigen (HBcAg)(18-27) CTL epitope and human immunodeficiency virus (HIV)-Tat(49-57) peptide was synthesized and the activity when adjuvanted with CpG oligodeoxynucleotide (CpG ODN) was evaluated in PBMCs from patients with chronic HBV infection in the immunotolerant phase in this study. Results showed that the fusion peptide when adjuvanted with CpG ODN could induce significantly higher levels of IFN-γ and IL-4 in the PBMCs compared with fusion peptide or CpG ODN alone. The magnitude of augmentation to IFN-γ by the fusion peptide plus CpG ODN was much higher than that to IL-4. Cytotoxicity assay showed that the percentage of target cell lysis by effector cells stimulated by fusion peptide plus CpG ODN was higher than that in fusion peptide or CpG ODN alone at most of the E/T ratios tested. The magnitude augmented to IFN-γ by fusion peptide plus CpG ODN was also much higher than that to the percentage of target cell lysis. It is concluded that HBcAg(18-27) and HIV-Tat(49-57) fusion peptide when adjuvanted with CpG ODN may have much higher potency to induce IFN-γ than to induce IL-4 and cytotoxicity, suggesting the favorable immune response towards noncytolytic inactivation of the virus mediated by IFN-γ and the potential to break the tolerant state in chronic HBV infection., (Copyright © 2010 Elsevier B.V. All rights reserved.)

In this issue of Clinical Cancer Research, Zoglmeier and colleagues show that CpG, via the induction of IFN-α, matures myeloid-derived suppressor cells to abrogate immune suppression in 2 murine solid tumor models.

Brucella abortus, a gram negative, facultative intracellular pathogen causes brucellosis in many animal species and humans. Although live, attenuated vaccines are available against this infection, they suffer from certain limitations. Therefore, the development of an effective subunit vaccine against brucellosis is an area of intense research. The outer membrane proteins (OMPs) of Brucella species have been extensively studied for its immunogenicity and protective ability. We have investigated the potential of CpG ODN to enhance the immunogenicity and protective efficacy of recombinant 28 kDa outer membrane protein (rOMP28) of Brucella melitensis. The study demonstrated vigorous immunoglobulin G (IgG) response of OMP28. The administration of rOMP28 with CpG caused increased cell mediated immune response in terms of induced IgG2a, T-cell proliferation and up-regulation of type I cytokine expression. In contrast, the free antigen suppressed the interferon gamma (type I cytokine) production on in-vitro stimulation of spleenocytes. The result indicates the role of OMP28 in the down regulation of IFN-gamma production. Moreover, the B. abortus S-19 vaccinated mice showed highest production of IL-4 and IFN-gamma. The protective ability of the antigen was evaluated by systemic bacterial clearance after challenging the mouse with B. abortus 544 pathogen. The level of protection was significant in rOMP28+CpG treated mice but was lower than the required level. The results of the present study indicate that rOMP28 could be an immunogen capable of inducing both humoral and cellular immune response. The humoral response was biased towards Th1 type when it was co-administered with CpG.

DNA damage by adduct formation is a critical step for the initiation of carcinogenesis. Aromatic amines are strong inducers of environmental carcinogenesis. Their DNA adducts are known to exist in an equilibrium between the major groove (B) and base-displaced stacked (S) conformations. However, the factors governing such heterogeneity remain unclear. Here we conducted extensive calorimetry/NMR/CD studies on the model DNA lesions caused by fluorinated 2-aminfluorene (FAF) and 4-aminobiphenyl (FABP) in order to gain thermodynamic and kinetic insights into the S/B conformational equilibrium. We demonstrate that there are large differences in enthalpy-entropy compensations for FABP and FAF. The small and flexible FABP exclusively adopts the less perturbed B conformer with small enthalpy (DeltaDeltaH-2.7 kcal/mol)/entropy (DeltaDeltaS-0.7 eu) change. In contrast, FAF stacks better and exists as a mixture of B and S conformers with large enthalpy (DeltaDeltaH-13.4 kcal/mol)/entropy (DeltaDeltaS-34.2 eu) compensation. van't Hoff analysis of dynamic (19)F NMR data indicated DeltaH(B<-->S) = 4.1 kcal/mol in favor of the B conformer and DeltaS(B<-->S) = 15.6 cal mol(-1) K(-1) in favor of the intercalated S conformer. These findings demonstrate that the favorable entropy of the S conformer over B conformer determines the S/B population ratios at physiological temperatures.

For decades, CFA has been the classic adjuvant for the induction of experimental autoimmune encephalomyelitis (EAE). Its encephalitogenic activity has been originally linked to the induction of Th1 responses. CpG, which is also a potent Th1 inducer, has been suggested by some studies to be comparably encephalitogenic. In this study, using the SJL proteolipid protein (PLP) 139-151 peptide EAE model, we show that active immunizations using CFA but not CpG 1826/IFA as an adjuvant induced disease. Passive induction of EAE resulted in severe disease when cells were transferred from PLP in CFA-primed mice but resulted in only a mild, transient disease when cells originated from PLP in CpG 1826/IFA-primed mice. In accordance with these findings, immunizations using CFA but not CpG 1826/IFA as an adjuvant elicited a delayed-type hypersensitivity response. ELISPOT analysis revealed that CFA promoted the differentiation of much higher levels of PLP-specific, IL-17-secreting cells compared with CpG 1826/IFA. Both adjuvants induced comparable frequencies of PLP-specific, IFN-gamma-secreting cells and also induced Ag-specific proliferation to the same extent. The severity of EAE in PLP in CFA-immunized mice was reduced when IL-17 was neutralized in vivo, demonstrating the crucial role of this cytokine in disease induction. The data show that immunizations using the autoantigen in CpG 1826/IFA result in very low frequencies of Ag-specific IL-17 cells, suggesting a lower risk of Th17-mediated pathology when using this adjuvant.

The bacterial Tn5 and Tn10 transposases have a single active site that cuts both strands of DNA at their respective transposon ends. This is achieved using a hairpin intermediate that requires the DNA to change conformation during the reaction. In Tn5 these changes are controlled in part by a flipped nucleoside that is stacked on a tryptophan residue in a hydrophobic pocket of the transposase. Here we have investigated the base flipping mechanism in Tn10 transposition. As in Tn5 transposition, we find that base flipping takes place after the first nick and is required for efficient hairpin formation and resolution. Experiments with an abasic substrate show that the role of base flipping in hairpin formation is to remove the base from the DNA helix. Specific interactions between the flipped base and the stacking tryptophan residue are required for hairpin resolution later in the reaction. We show that base flipping in Tn10 transposition is not a passive reaction in which a spontaneously flipped base is captured and retained by the protein. Rather, it is driven in part by a methionine probe residue that helps to force the flipped base from the base stack. Overall, it appears that base flipping in Tn10 transposition is similar to that in Tn5 transposition.

In this report, we investigated the adjuvant effect of CpG-ODN on the immunogenicity and protective efficacy of influenza M2e peptide vaccine. We found that the addition of CpG-ODN 1826 into aluminum-adjuvant M2e peptide vaccine increased M2e-specific Th1 immune response, indicated by higher titers of M2e-specific IgG2a and more IFN-gamma-secreting lymphocytes. However, according to the result from virus challenge, enhancement of M2e-specific Th1 immune response failed to increase the protection against influenza virus. Moreover, when challenged with high dose of influenza virus, the addition of CpG-ODN even weakened the protective activity. These results suggested that the intensity of immune responses was not simply correlated with protective activity of influenza M2e vaccine and more comprehensive criterion should be built up for the evaluation of M2e-based vaccine.

The need to enhance the immunogenicity of purified subunit antigens and modulate resulting immune responses has prompted the development of new adjuvants. Here, the ability of CpG oligodeoxynucleotides (ODN), a bovine host defence peptide indolicidin, and polyphosphazene to synergistically combine and enhance innate and adaptive immune responses was examined in mice. In vitro, the adjuvant combination of CpG ODN, indolicidin and polyphosphazene (CpG/indol/PP) enhanced the secretion of TNF-alpha, IL-12p40, and IL-6 by bone marrow-derived DCs (BMDCs) when compared to the individual components. When co-formulated with ovalbumin (OVA), CpG/indol/PP formed antigen-adjuvant complexes, and enhanced antibody and cell-mediated responses in mice, via both MHC I and II pathways, promoting a more balanced antibody-mediated and type 1-biased cell-mediated immune response. Furthermore, substitution of the proline residues of indolicidin with arginine increased the synergistic adjuvant effect of the peptide, and induced significantly higher IgG1 and IgG2a titers and IFN-gamma secretion, as well as increased uptake by antigen presenting cells. These results clearly demonstrate that the use of a combination of CpG ODN, indolicidin, and polyphosphazene as adjuvant can significantly enhance an antigen-specific immune response.

Unmethylated CpG oligodeoxynucleotides (ODNs), by activating cells of the innate immune system, such as dendritic cells and NK cells, are potent adjuvants for type 1 immune responses. In the present study, we aimed to investigate the role of invariant NKT (iNKT) cells, a subset of lipid-reactive innate lymphocytes, in CpG ODN-induced innate and acquired type 1 responses. Our data show that, in response to the CpG ODN type B 1826, splenic and hepatic iNKT cells become activated and produce IFN-gamma, but not IL-4, both in vitro and in vivo. This Th1 bias is independent from the Ag-presenting molecule CD1d and strongly requires IL-12, at least in vitro. We also report that iNKT cell activation, in response to CpG ODN type B, results in the transactivation of NK cells. To address the potential role of iNKT cells in type 1 innate immunity induced by CpG ODN, a murine model of malignant melanoma was used. We show that CpG ODN type B protects mice against B16F10-induced lung metastasis in wild-type mice, but in a less efficient manner in iNKT cell-deficient animals. Finally, we report that immunization of wild-type mice with CpG ODN type B plus keyhole limpet hemocyanin biases the immune response toward a Th1 direction, an effect strongly mediated by iNKT cells. We conclude that iNKT cells amplify the innate and acquired response to CpG ODN type B, with potentially important consequences for the regulation of immune responses.

8-Oxo-7,8-dihydro-2'-deoxyguanosine (OG), a prevalent product of oxidative stress on cellular DNA, is readily further oxidized forming adducts with nucleophiles. In the presence of tyrosine or p-cresol, an unusual tricyclo[4.3.3.0] adduct has been characterized in both nucleoside and oligodeoxynucleotide studies. The adduct is more stable in oligomers than nucleosides and undergoes slow reversion and hydration to spiroiminodihydantoin.

We demonstrate, for the first time, a robust novel polydimethylsiloxane (PDMS) chip that can provide controlled pulsatile release of DNA based molecules, proteins and oligonucleotides without external stimuli or triggers. The PDMS chip with arrays of wells was constructed by replica molding. Poly(lactic acid-co-glycolic acid) (PLGA) polymer films of varying composition and thickness were used as seals to the wells. The composition, molecular weight and thickness of the PLGA films were all parameters used to control the degradation rate of the seals and therefore the release profiles. Degradation of the films followed the PLGA composition order of 50:50 PLGA>75:25 PLGA>85:15 PLGA at all time-points beyond week 1. Scanning electron microscopy images showed that films were initially smooth, became porous and ruptured as the osmotic pressure pushed the degrading PLGA film outwards. Pulsatile release of DNA was controlled by the composition and thickness of the PLGA used to seal the well. Transfection experiments in a model Human Embryonic Kidney 293 (HEK293) cell line showed that plasmid DNA loaded in the wells was functional after pulsatile release in comparison to control plasmid DNA at all time-points. Thicker films degraded faster than thinner films and could be used to fine-tune the release of DNA over day length periods. Finally the PDMS chip was shown to provide repeated sequential release of CpG oligonucleotides and a model antigen, Ovalbumin (OVA), indicating significant potential for this device for vaccinations or applications that require defined complex release patterns of a variety of chemicals, drugs and biomolecules.

CpG are powerful drugs activating the innate immune system. In this study, the ability of their intramammary administration in impeding the devastating progression of carcinogenesis in all the mammary glands of female BALB/c mice transgenic for the rat neu transforming oncogene was assessed. Starting when in situ carcinomas were scattered over all their mammary glands (week 10), mice received CpG injections in the stroma of the fourth left gland. Local neoplastic progression was inhibited by six monthly administrations. CpG not only delayed the onset of carcinomas in the injected gland, but also hampered their progression. Extended latency was observed for tumors in glands both close to and far from the injection site. When the experiment ended (week 45), no tumors were palpable in 67% of the injected glands and a markedly impaired tumor growth was evident in the others. An impressive local infiltrate of CD11b(+) cells with the morphologic features of macrophages, plasma cells, B220(+) B cells, and CD4(+) and CD8(+) T cells was quickly recruited to the CpG-treated glands. High quantities of IFN-gamma producing cells were only present in the ipsilateral axillary draining lymph nodes of the treated glands. Enhanced natural killer (NK) lytic activity was also detected in the spleens. Inhibition of progression was weaker when only four injections were given, and abolished by in vivo depletion of NK cells. CpG monotherapy is thus effective in an aggressive model of autochthonous cancer. The results strongly support the administration of CpG as a local monotherapy of multiple invasive microscopic lesions.

We introduce a method for the bottom-up assembly of biomolecular structures that combines the precision of the atomic force microscope (AFM) with the selectivity of DNA hybridization. Functional units coupled to DNA oligomers were picked up from a depot area by means of a complementary DNA strand bound to an AFM tip. These units were transferred to and deposited on a target area to create basic geometrical structures, assembled from units with different functions. Each of these cut-and-paste events was characterized by single-molecule force spectroscopy and single-molecule fluorescence microscopy. Transport and deposition of more than 5000 units were achieved, with less than 10% loss in transfer efficiency.

CpG oligodeoxynucleotides (ODN) stimulate the immune system and are under evaluation as treatments and vaccine adjuvants for infectious diseases, cancer, and immune system disorders. Although they have shown promising results in numerous clinical trials, the ultimate use of CpG ODN-based therapeutics may hinge on improved pharmacokinetics and reduced systemic side-effects. CpG ODN efficacy and potency might be enhanced greatly by packaging them into particles that protect them from degradation and specifically target them for uptake by immune-competent cells. The plasma proteinase inhibitor alpha 2-macroglobulin (alpha 2M) binds numerous biologically active macromolecules, including cytokines, chemokines, and growth factors, and can modulate their activity. Molecules bound to alpha 2M are protected from interactions with neighboring macromolecules and are targeted for receptor-mediated uptake by immune-competent cells. Here, we report that activated alpha 2M (alpha 2M*) binds CpG ODN and enhances their immunostimulatory properties significantly. Murine macrophages treated with alpha 2M*-ODN complexes respond more rapidly and produce a greater cytokine response than induced by free CpG ODN. Using human PBMC, alpha 2M*-ODN complexes exhibit fourfold enhanced potency and 15-fold greater efficacy for stimulating production of inflammatory cytokines. alpha 2M* targets delivery of CpG ODN specifically to immune-competent cells, which endocytose the complexes sixfold more rapidly than free CpG ODN. CpG ODN bound to alpha 2M* are also protected from degradation by nucleases. This novel targeting technology may improve CpG ODN-based therapeutics by increasing efficacy at reduced doses, thus reducing side-effects and cost.

Molecular analyses of single or very few cells are often handicapped by low amounts of DNA or RNA from starting material. Specimens like biopsies, embryonic stem cells, or laser-microdissected tissues often do not provide nucleic acid quantities required for robust amplification and verification by diagnostic polymerase chain reaction (PCR)-based analyses. A simultaneous isolation procedure for both RNA and DNA from a single sample would greatly facilitate demanding molecular studies. Here, we describe a new method that combines the isolation of mRNA using commercially available oligo (dT)-bound magnetic beads with the precipitation of DNA from the remaining cell lysate. Both types of nucleic acids can then be separately preamplified by improved primer extension preamplification and subsequently used for multiple molecular analyses. With this procedure we were able to reliably gain DNA for PCR from as few as 5 cells. RNA suitable for various reverse transcription-PCR approaches and sequencing analyses could simultaneously be isolated even from single cell preparations.

Topical application of adjuvant to the skin promotes the generation of immune responses to co-administered peptide or protein antigen. We demonstrate that topical administration of CpG adjuvant (a TLR9 agonist) induces the cross-presentation of, and antigen-specific CTL induction to, locally injected soluble protein antigen. C57BL/6 mice were immunized by subcutaneous or intramuscular injection with ovalbumin (OVA) protein as model antigen. Application of CpG to the local skin induced more efficient cross-presentation of the injected antigen than co-injected adjuvant. Robust antigen-specific CTL responses were generated, as determined by antigen-specific CTL enumeration using tetramers, IFN-gamma ELISPOT analysis and cytotoxicity assays. Long-term memory CTL responses were induced. Topical administration of adjuvant induced Langerhans cell migration, local type 1 IFN-dependent myxovirus-resistance protein A expression and bystander dendritic cell (DC) activation. Soluble antigen-bearing DC within the skin draining lymph nodes were mainly CD11chiCD11bhilangerinloDEC205lo. Topical administration did not result in the splenomegaly or systemic cytokine induction (including TNF-alpha, IL-12, IFN-gamma and MCP-1) noted with parenteral administration. Topical TLR9 family agonists may be used to modulate the immune response to soluble protein vaccines administered by standard percutaneous route. Topical adjuvant administration increases efficacy of CTL induction and reduces toxicity when compared to parenteral adjuvant administration.

CpG ODN stimulates a TH1 response through its receptor Toll-like receptor 9 (TLR9). TLR9 is a receptor that is found intracellularly. Microparticles are efficiently internalized by dendritic cells (DCs) and macrophages and would thus be an ideal delivery vehicle for CpG ODN to reach its target site thereby enhancing the TH1 response to an antigen also encapsulated in the microparticle. Here, we show that careful control over fabrication parameters can produce biodegradable microparticles with predictable size distributions, surface morphology, and shape. Entrapment efficiencies of the model antigen OVA ranged from 19% to 23% with an average loading of 10 microg/mg of microparticles. For CpG ODN, these values were 33% to 35%, which corresponded to an average loading of 8.5 microg/mg of microparticles. The microparticles release CpG ODN and OVA in a burst followed by sustained release profile. At the highest concentration of microparticles incubated with a pure DC cell line, 92% of DCs had internalized microparticles by 16 hours, confirming that DCs efficiently take up the microparticles. Microparticles are capable of inducing DC maturation as determined by up-regulation of CD80 and CD86 markers. Although the presence of CpG ODN in the microparticles did not impact on the phenotype of the DCs, it was necessary for DCs to induce activation of antigen-specific T cells as indicated by interferon-gamma production. Microparticles entrapping both antigen and CpG ODN induced significantly higher amounts of anti-OVA antibody production than other preparations such as the soluble OVA and CpG ODN (P<0.01) and stimulated stronger IgG2a production than delivery of microparticles entrapping antigen alone. We conclude that co-encapsulating immunostimulatory CpG ODN and antigen in degradable microparticles is an effective approach to enhancing development of a TH1 immune response.

Prion diseases are characterized by conversion of the cellular prion protein (PrP(C)) to a protease-resistant conformer, the srapie form of PrP (PrP(Sc)). Humoral immune responses to nondenatured forms of PrP(Sc) have never been fully characterized. We investigated whether production of antibodies to PrP(Sc) could occur in PrP null (Prnp(-/-)) mice and further, whether innate immune stimulation with the TLR9 agonist CpG oligodeoxynucleotide (ODN) 1826 could enhance this process. Whether such stimulation could raise anti-PrP(Sc) antibody levels in wild-type (Prnp(+/+)) mice was also investigated. Prnp(-/-) and Prnp(+/+) mice were immunized with nondenatured 139A scrapie-associated fibrils (SAF), with or without ODN 1826, and were tested for titers of PrP-specific antibodies. In Prnp(-/-) mice, inclusion of ODN 1826 in the immunization regime increased anti-PrP titers more than 13-fold after two immunizations and induced, among others, antibodies to an N-terminal epitope, which were only present in the immune repertoire of mice receiving ODN 1826. mAb 6D11, derived from such a mouse, reacts with the N-terminal epitope QWNK in native and denatured forms of PrP(Sc) and recombinant PrP and exhibits a K(d) in the 10(-)(11) M range. In Prnp(+/+) mice, ODN 1826 increased anti-PrP levels as much as 84% after a single immunization. Thus, ODN 1826 potentiates adaptive immune responses to PrP(Sc) in 139A SAF-immunized mice. These results represent the first characterization of humoral immune responses to nondenatured, infectious PrP(Sc) and suggest methods for optimizing the generation of mAbs to PrP(Sc), many of which could be used for diagnosis and treatment of prion diseases.

Biological organisms perform complex information processing and control tasks using sophisticated biochemical circuits, yet the engineering of such circuits remains ineffective compared with that of electronic circuits. To systematically create complex yet reliable circuits, electrical engineers use digital logic, wherein gates and subcircuits are composed modularly and signal restoration prevents signal degradation. We report the design and experimental implementation of DNA-based digital logic circuits. We demonstrate AND, OR, and NOT gates, signal restoration, amplification, feedback, and cascading. Gate design and circuit construction is modular. The gates use single-stranded nucleic acids as inputs and outputs, and the mechanism relies exclusively on sequence recognition and strand displacement. Biological nucleic acids such as microRNAs can serve as inputs, suggesting applications in biotechnology and bioengineering.

With the goal to design ligands recognizing extended regions on dsDNA, a covalent dimer of the fluorescent dye Hoechst 33258 [bis-HT(NMe)] composed of two dye molecules linked via the phenol oxygen atoms with a (CH2)3-N+ H(CH3)-(CH2)3 fragment was constructed using computer modeling and then synthesized. Its interactions with the double-stranded DNA (dsDNA) were studied by fluorescent and UV-Vis spectroscopy and circular (CD) and linear dichroism (LD). Based on variations in the affinity to the dsDNA, it was shown that complexes of three types are formed. The first type complexes result from binding of a bis-HT(NMe) monomer in the open conformation; in this case the ligand covers the total dsDNA turn and is located in the minor groove according to the positive value of CD at 370 nm. In addition, the ability to form bis-HT(NMe)-bridges between two dsDNA molecules, i.e., each of the two bis-HT(NMe) ends binds to two different dsDNA molecules, was demonstrated for the first type complexes. Spectral characteristics (maximal absorption at 362 nm, positive sign, and maximal value of CD at 370 nm) of the first type complexes conform to those of the specific Hoechst 33258 complex with poly[d(A-T)] x poly[d(A-T]. The second type complexes correspond to the bis-HT(NMe) sandwich (as an inter- or intramolecular) binding to dsDNA with stoichiometry > or = 5 bp. Thereby, a negative LD at 360 nm and the location of bis-HT(NMe) sandwiches in the minor groove of B form dsDNA seems contradictory. Spectral characteristics (maximal positive CD at 345 nm, a dramatic decrease in fluorescence intensity and the shift of its maximum to 490 nm) of these complexes favor a suggestion that this binding correlates to the formation of nonspecific dimeric Hoechst 33258 complex with dsDNA. The third type complexes are characterized by stoichiometry of one bis-HT(NMe) molecule per approximately 2 bp and the tendency to zero of LD values at 270 and 360 nm. We assume that in these complexes bis-HT(NMe) sandwich dimers are formed on dsDNA. The complexes of this type conform to the aggregation type complex of Hoechst 33258 with dsDNA. The ability of bis-HT(NMe) to cover the whole dsDNA turn or form bridges with two dsDNA upon the formation of the first type complexes essentially distinguishes it from Hoechst 33258, which can only occupy 5 bp and does not form such bridges. This specific property of bis-HT(NMe) may support new biological activities.

Natural killer dendritic cells (NKDC) are a novel subtype of dendritic cells with natural killer (NK) cell properties. IFN-gamma is a pleiotropic cytokine that plays an important role in the innate immune response to tumors. Based on our previous finding that the combination of Toll-like receptor 9 ligand CpG and interleukin (IL)-4 stimulates NKDC to produce IFN-gamma, we hypothesized that NKDC are the major IFN-gamma-producing dendritic cell subtype and may play a significant role in the host antitumor response. We found that under several conditions in vitro and in vivo NKDC accounted for the majority of IFN-gamma production by murine spleen CD11c(+) cells. IL-18 alone induced NKDC to secrete IFN-gamma, and the combination of IL-18 and CpG resulted in a synergistic increase in IFN-gamma production, both in vitro and in vivo. NK cells made 26-fold less IFN-gamma under the same conditions in vitro, whereas dendritic cells produced a negligible amount. The mechanism of IFN-gamma secretion by NKDC depended on IL-12. NKDC selectively proliferated in vitro and in vivo in response to the combination of IL-18 and CpG. Systemic treatment with IL-18 and CpG reduced the number of B16F10 melanoma lung metastases. The mechanism depended on NK1.1(+) cells, as their depletion abrogated the effect. IL-18 and CpG activated NKDC provided greater tumor protection than NK cells in IFN-gamma(-/-) mice. Thus, NKDC are the major dendritic cell subtype to produce IFN-gamma. The combined use of IL-18 and CpG is a viable strategy to potentiate the antitumor function of NKDC.

Background and Purpose: Synthetic oligodeoxynucleotides (ODNs) containing unmethylated cytosine-guanine (CpG) motifs bind to Toll-like receptor 9 (TLR9) and stimulate both innate and adaptive immune reactions and possess anti-tumor activity. We recently reported that CpG ODN 1826 strongly enhances radioresponse of both immunogenic [Milas L, Mason K, Ariga H, et al. CpG oligodeoxynucleotide enhances tumor response to radiation. Cancer Res 2004;64:5074-7] and non-immunogenic [Mason KA, Ariga H, Neal R, et al. Targeting toll-like receptor-9 with CpG oligodeoxynucleotides enhances tumor response to fractionated radiotherapy. Clin Cancer Res 2005;11:361-9] murine tumors. Using two immunogenic murine tumors, a fibrosarcoma (FSa) and a mammary carcinoma (MCa-K), the present study explored whether CpG ODN 1826 also improves the response of murine tumors to the chemotherapeutic agent docetaxel (DOC)., Materials and Methods: CpG ODN 1826 (100 microg) was given sc three times: when leg tumors were 6mm, when they grew to 8mm and again 1 week later. DOC (33 mg/kg iv) and local tumor radiation (10Gy) were given when tumors were 8mm. Effects of the treatments were assayed by tumor growth delay, defined as days for tumors to grow from 8 to 12 mm in diameter., Results: Treatment with CpG ODN 1826 resulted in strongly enhanced response of FSa tumors to radiation and MCa-K tumors to the chemotherapeutic agent DOC. Enhancement of tumor treatment response was demonstrated by a strong prolongation in the primary tumor treatment endpoint, tumor growth delay. Coincidentally, this treatment also resulted in a higher rate of tumor cure than that observed after tumor radiotherapy or chemotherapy alone. When all three agents were combined the effect was comparable to that of the combination of CpG ODN 1826 with radiation in the case of FSa or of the combination of CpG ODN 1826 with DOC in the case of MCa-K., Conclusion: Overall results show that CpG ODN 1826 can markedly improve tumor response to radiation and chemotherapy (DOC), suggesting that CpG ODNs have potential to be beneficial when used singly or in combination with other standard treatment modalities such as taxane chemotherapy, radiotherapy or both.

The p53 tumor suppressor protein binds to DNA as a dimer of dimers to regulate transcription of genes that mediate responses to cellular stress. We have prepared a cross-linked trapped p53 core domain dimer bound to decamer DNA and have determined its structure by x-ray crystallography to 2.3A resolution. The p53 core domain subunits bind nearly symmetrically to opposite faces of the DNA in a head-to-head fashion with a loophelix motif making sequence-specific DNA contacts and bending the DNA by about 20 degrees at the site of protein dimerization. Protein subunit interactions occur over the central DNA minor groove and involve residues from a zinc-binding region. Analysis of tumor derived p53 mutations reveals that the dimerization interface represents a third hot spot for mutation that also includes residues associated with DNA contact and protein stability. Residues associated with p53 dimer formation on DNA are poorly conserved in the p63 and p73 paralogs, possibly contributing to their functional differences. We have used the dimeric protein-DNA complex to model a dimer of p53 dimers bound to icosamer DNA that is consistent with solution bending data and suggests that p53 core domain dimer-dimer contacts are less frequently mutated in human cancer than intra-dimer contacts.

Low-dose CpG ODN pretreatment is known to induce effective protective immunity against acute infectious diseases. In the present study, using primary murine peritoneal macrophages and the macrophage-like cell line, RAW264.7, we investigated whether low-dose CpG ODN pretreatment would induce hyporesponsiveness in response to a subsequent high-dose CpG ODN challenge and further investigated the molecular mechanisms underlying this event. Our results showed that pretreatment with a low dose of CpG ODN inhibits TNF-alpha production stimulated by later high-dose CpG ODN stimulation in a dose- and time-dependent manner. Interestingly, anti-mouse TLR9 blocking antibody added prior to CpG ODN pretreatment did not affect TNF-alpha release, but antibody added after CpG ODN pretreatment augmented the pretreatment effect of CpG ODN. This difference suggests that cell-surface TLR9 is indeed functional on activated cells. Flow cytometry revealed that low-dose CpG ODN pretreatment decreased cell-surface binding and internalization of a subsequent high-dose stimulation, suggesting that decreased internalization of succeeding CpG ODN is associated with reduced TNF-alpha release. Although both intracellular and cell-surface TLR9 expression are observed, low dose of CpG ODN pretreatment increased only cell-surface TLR9 levels. Importantly, low-dose CpG ODN pretreatment also significantly inhibited the activation of NF-kappaB, an important downstream regulator of various proinflammatory cytokines. In summary, our results demonstrate that suppression of TNF-alpha production by low dose of CpG ODN pretreatment correlates with decreased binding and internalization of subsequent CpG ODN, decreased intracellular content of TLR9, and inhibition of NF-kappaB activation.

Background/aims: The high rate of local recurrence after radical resection of pancreatic ductal adenocarcinoma fosters intensive efforts to develop new approaches for adjuvant treatment. Antisense and modified oligodeoxynucleotides (ODNs) have demonstrated significant tumour growth inhibitory effects in preclinical systems. Our aim was to evaluate the possible therapeutic potential of ODNs containing unmethylated deoxycytidyl-deoxyguanosine dinucleotides (CpG motifs)., Methodology: For in vitro analysis, [(3)H]thymidine incorporation for DNA synthesis, colorimetric cell vitality assay (EZ4U assay), and DNA fragmentation assay (JAM-[(3)H]thymidine incorporation assay) to test for apoptosis were performed. In vivo testing was done on an orthotopic pancreatic xenotransplantation model using severe combined immunodeficient (SCID) beige and nude mice., Results: No significant differential effect of either control ODN or CpG-1826 ODN with regard to tumour cell proliferation or induction of apoptosis was observed in vitro. In vivo, ODN-1826 proved to have a significant inhibitory effect (up to 40% reduction of tumour weight) when compared with tumour-bearing animals treated with saline or control ODN. This was accompanied by sevenfold increase in splenomegaly and moderate hepatomegaly. The reduction of tumour weight by ODN-1826 was only slightly more pronounced in nude compared with SCID beige mice., Conclusion: CpG-1826 induces significant growth-inhibitory effects on orthotopic xenotransplanted pancreatic tumours in highly immunodeficient mice, which might be explained by innate immunity mechanisms and possibly a complex interaction of tumour and stroma cells.

Immunogenicity of tumor cells is generally weak. Therefore, dendritic cells (DCs) have been used to boost anti-tumor responses of DC-based vaccines. DC function is highly dependent on its subsets and the level of its maturation. Nowadays, DC/tumor cell fusion vaccines are already used in clinical trials, and there are numerous studies discussing the effects of cytidine-phosphate-guanosine-containing oligonucleotides (CpG-ODN) on various cell types including DC. CpG-ODN a powerful immuno-stimulant can drive DCs fully mature, thus improve the efficacy of vaccine therapy. There are two simple ways to help load tumor antigens onto DCs by direct contact with cells themselves: fusion or co-culture of DCs with whole tumor cells. In this study, we combined these two approaches to improve the efficacy of DC/tumor cell-based vaccine. Mature DCs are adept at presenting processed Ag to T cells with loss of its capacity to capture Ag, while immature DCs are on the contrary. Our results emphasize the necessity of considering the stage of DC maturation and corresponding choice of tumor antigen delivery when designing approaches for prophylaxis or therapy of tumors using DC-based immunization protocols. We used CpG-ODN-1826-stimulated mature DCs and non-CpG-ODN-stimulating DCs as sources of tumor antigen carriers to investigate the appropriate Ag-loading ways between fusion and co-culture. Our results displayed that DC/tumor vaccine using CpG-ODN-stimulating mature DCs fused, not co-cultured, with tumor cells can generate a consistent and highly effective anti-tumor immune responses in vivo.

The binding of manganese(III)-tetra(4-N-methylpyridyl)porphyrin (MnTMpyP) with synthetic poly(dA-dT)2, poly(dI-dC)2, and poly(dG-dC)2 DNAs as well as calf thymus (CT) DNA has been quantitatively studied in detail using induced CD (circular dichroism) spectroscopy in the Soret absorption band. The CD spectra, which changed greatly depending on the porphyrin to DNA base-pair molar ratio (r), were normalized with respect to DNA concentration and deconvoluted. Three independent component binding modes (named mode 1, 2, and 3 in the order of increasing r values) were identified, which successfully simulated the observed CD spectra with negligibly small residuals for a wide range of r values. In the case of poly(dA-dT)2, poly (dI-dC)2, and CT DNA, all the three modes appeared, whereas in the case of poly(dG-dC)2 DNA, only modes 1 and 3 appeared in the r range studied. The r dependence of each binding mode, i.e., its relative affinity toward DNA, has been revealed by this analysis. Mode 1, which appeared as a single binding mode at very low r values (r < or = ca. 0.05), was inhibited by the addition of methyl green, a drug that preferentially binds to the major groove of poly (dA-dT)2 DNA. Berenil, a known minor groove binder to poly(dA-dT)2 or poly(dI-dC)2 DNA, inhibited modes 2 and 3. From these inhibition experiments as well as comparison of the component spectra for DNAs of different sequence, a binding site on DNA was proposed for each component binding mode. The number of DNA base pairs covered by a single molecule of porphyrin was estimated., (Copyright 2005 Wiley Periodicals, Inc.)

Burkholderia pseudomallei, the causative agent of melioidosis, is a gram-negative bacterium which can cause either chronic infections or acute lethal sepsis in infected individuals. The disease is endemic in Southeast Asia and northern Australia, but little is known about the mechanisms of protective immunity to the bacterium. In this study, we have developed a procedure to utilize dendritic cells in combination with CpG oligodeoxynucleotides as a vaccine delivery vector to induce protective immune responses to various strains of B. pseudomallei. Our results show that strong cell-mediated immune responses were generated, while antibody responses, although low, were detectable. Upon virulent challenge with B. pseudomallei strain K96243, NCTC 4845, or 576, animals immunized with dendritic cells that were pulsed with heat-killed K96243 and matured in the presence of CpG 1826 showed significant levels of protection. These results show that a vaccine strategy that actively targets dendritic cells can evoke protective immune responses.

Type 1 diabetes is considered as Th1 cell mediated autoimmune disease and the suppression of Th1 cells or the activation of Th2 cells has been regarded as a plausible immunologic intervention for the prevention of type 1 diabetogenesis in a rodent model. CpG ODN is an immunostimulatory sequence primarily present in bacterial DNA, viral DNA and BCG. CpG ODN is conventionally classified as a Th1 cell activator, which has been clinically applied to cancer, allergy and infectious disease. Recently, there was a promising report of that CpG ODN administration suppressed the development of type 1 diabetes in NOD mice by inducing Th2 cell mediated cytokine. However, the antidiabetogenic effect of CpG ODN on NOD mice is controversial. Thus, two studies were serially undertaken with various kinds of CpG motif to find a more optimal sequence and administration method. In the first study, CpG ODN was vaccinated four times and pancreatic inflammation and the quantity of serum insulin subsequently evaluated. In the second study, the amounts of IFN gamma and IL-4 in sera were measured as representative cytokines of Th1 and Th2 cells, respectively. As a result, vaccination or continuous injection of CpG ODN failed to show a preventive effect on type 1 diabetogenesis in NOD mice. Structural differences of CpG ODN also had no affect on the result. CpG ODN also consistently showed affect on the pancreatic pathology. The productions of IFN gamma and IL-4 were detected only in the K and D type CpG ODN administration groups. Comparison of the two cytokines leads to the conclusion that CpG ODN generated a Th1-weighted response in both study groups. It was assumed that CpG ODN failed to produce Th2-weighted cytokine milieu, which can overcome the genetically determined phenotype of NOD mice. Given these results, it was concluded that the immunotherapeutic application of CpG ODN on Type 1 diabetes had clear limitations.

The HER-2 gene is overexpressed in a subset of breast, ovarian, lung, and pancreatic cancers. Antisense oligonucleotides suppress gene expression depending on the stability of the DNA.RNA hybrids formed at the target site. Polyamines, the cellular cations that interact with DNA and RNA, may influence hybrid stability in the cell. Therefore, we studied the ability of natural polyamines (putrescine, spermidine, and spermine) and a series of their structural analogues to stabilize DNA.RNA and RNA.RNA duplexes using melting temperature (T(m)) measurements and circular dichroism (CD) spectroscopy. Phosphodiester (PO) and phosphorothioate (PS) oligonucleotides (ODNs) (15 nucleotides, 5'-CTCCATGGTGCTCAC-3') targeted to the initiation codon region of the HER-2 mRNA, and complementary RNA and DNA ODNs, were used in this study. The relative order of thermal stability was as follows: RNA.RNA > PO-DNA.RNA > PO-DNA.PO-DNA > PS-DNA.RNA > PS-DNA.PO-DNA > PS-DNA.PS-DNA. The ability of polyamines to stabilize the duplexes improved with the cationicity of the polyamine, with hexamines being more effective than pentamines, which in turn were more effective than tetramines and triamines. However, chemical structural effects were clearly evident with isovalent homologues of spermidine and spermine. CD spectra showed B and A conformations, respectively, for the DNA and RNA helices. DNA.RNA hybrids adopted an intermediate structure between the B and A forms. These data help us to understand the role of endogenous polyamines in DNA.RNA hybrid stabilization, and provide information for designing novel polyamines to facilitate the use of antisense ODNs for controlling HER-2 gene expression.

The greatest biomedical challenge of this century is to develop a preventive vaccine against Human Immunodeficiency Virus (HIV-1). For an HIV vaccine to be effective, it appears logical to develop new strategies that enhance the level of the immune response as well as steer it towards the desirable cellular type. In view of this, there is a need for rational inclusion of biological adjuvants into the HIV-1 vaccination strategies that could potentiate the immune responses both qualitatively and quantitatively. The adjuvant may include the immunostimulatory oligonucleotides containing CpG motifs, whose immunomodulatory characters are well established and represent the basis for an effective vaccine adjuvant. In our study, we investigated the use of an immunostimulatory oligonucleotide (or CpG motif), 1826-ODN to augment the immune response elicited by plasmid DNA vaccine constructs containing Indian subtype C HIV-1 envelope gp120 and gag-protease genes in Balb/c mouse model system. A dose of 2-microg CpG motifs/mouse was found to be optimum when co-administered with the DNA vaccine constructs with the peak level of humoral and cell mediated immune responses at 6 weeks post immunization. Murine IFN-gamma ELISpot assay demonstrated that the use of 1826-ODN led to a broad based and long term recognition of the subtype C envelope and gag peptides. The use of CpG motifs has been effective in augmenting the immune responses generated by the DNA constructs. Taken together, these results are an important advancement towards the design of future preclinical and clinical trials of these vaccine constructs.

DNA dodecamers modified with nucleotide building blocks based on a bicyclo[3. 1.0]hexane system that effectively locks the ribose template into an RNA-like or North (N) conformation were analyzed by various biophysical techniques including high field nuclear magnetic resonance (NMR). Replacement of either one or both of the center thymidines in the Dickerson Drew dodecamer (CGCGAAT*T*CGCG) caused a progressive shift in the bending propensity of the double helix as shown by a newly developed rapid technique that compares the residual dipolar coupling (RDC) values of the modified duplexes with those previously determined for the native DNA.

The major aim of this paper was to determine whether cationic microspheres (CM), consisting of the permeable polymer Eudragit RS 100 plus the cationic surfactant dioctadecyl-dimethyl-ammonium bromide (DDAB(18)), could bind to double-stranded peptide nucleic acid PNA-DNA-PNA (PDP) chimeras exhibiting decoy activity against NF-kappaB transcription factors. Microspheres were produced by the 'solvent evaporation method' and centrifugation at 500, 1,000 and 3,000 rpm to obtain different-sized microparticles. Microsphere morphology, size and size distribution were determined by optical and electron microscopy observations. In order to determine their binding activity, double-stranded DNA-based and PDP-based decoy molecules were incubated with different amounts of microparticles in the presence of 100 ng of either (32)P-labeled DNA-DNA or DNA-PDP hybrid molecules or cold PDP-PDP hybrids. The complexes were analyzed by agarose gel electrophoresis. The resistance of (32)P-labeled DNA-DNA and DNA-PDP molecules in the presence of serum or cellular extracts was evaluated after binding to CM by gel electrophoresis analysis. DDAB(18) Eudragit RS 100 microspheres are able to bind to DNA-PDP and PDP-PDP hybrids, to deliver these molecules to target cells and to protect DNA-PDP molecules from enzymatic degradation in simulated biological fluids. In addition, when assayed in ex vivo conditions, DDAB(18) Eudragit RS 100 microspheres exhibited low toxicity. The results presented in this paper demonstrate that CM can be considered suitable formulations for pharmacogenomic therapy employing double-stranded PDP chimeras.

Purpose: The effectiveness of interleukin (IL)-12-secreting tumor vaccines in the treatment of mouse tumors could be enhanced by concurrent application of cytokines and costimulatory molecules. We investigated the therapeutic potential of IL-12 gene-transduced melanoma vaccine in combination with CpG immunostimulatory oligodeoxynucleotide (ODN) 1826, an adjuvant known to favor development of Th1-biased immune response, in a B78-H1 (B78) melanoma model in mice., Experimental Design: Mice injected with B78 melanoma cells were treated with irradiated IL-12 gene-transduced B78 cells [B78/IL-12(X)] and/or ODN 1826. Mechanisms responsible for the antitumor effects of the treatment were investigated using fluorescence-activated cell sorter analysis, a standard (51)Cr releasing assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and ELISA., Results: Single injection of B78/IL-12(X) cells had no effect on tumor growth, whereas seven consecutive daily injections of ODN 1826 markedly inhibited tumor progression with occasional curative effects. When used in combination, B78/IL-12(X) cells and ODN 1826 caused additional tumor growth reduction and eradication of tumors in 62% of treated mice. The combined treatment activated local inflammatory response against tumor but also induced systemic antitumor immunity. In vitro studies have shown that when used together, B78/IL-12(X) cells and ODN 1826 induced a potent Th1 response and suggested the role of IFN-gamma in activation of the host immune response. The antitumor effects in double-treated mice were accompanied by the development of cytotoxic effectors in the spleen and activation of macrophages., Conclusions: The results provided the evidence that the combination of IL-12 gene-modified melanoma vaccine and ODN 1826 induces synergistically systemic and local antitumor immunity.

A software algorithm has been developed to investigate the folding process in B-DNA structures in vacuum under a simple and accurate force field. This algorithm models linear double stranded B-DNA sequences based on a local, sequential minimization procedure. The original B-DNA structures were modeled using initial nucleotide structures taken from the Brookhaven database. The models contain information at the atomic level allowing one to investigate as accurately as possible the structure and characteristics of the resulting DNA structures. A variety of DNA sequences and sizes were investigated containing coding and non-coding, random and real, homogeneous or heterogeneous sequences in the range of 2 to 40 base pairs. The force field contains terms such as angle bend, Lennard-Jones, electrostatic interactions and hydrogen bonding which are set up using the Dreiding II force field and defined to account for the helical parameters such as twist, tilt and rise. A close comparison was made between this local minimization algorithm and a global one (previously published) in order to find out advantages and disadvantages of the different methods. From the comparison, this algorithm gives better and faster results than the previous method, allowing one to minimize larger DNA segments. DNA segments with a length of 40 bases need approximately 4 h, while 2.5 weeks are needed with the previous method. After each minimization the angles between phosphate-oxygen-carbon A1, the oxygen-phosphate-oxygen A2 and the average helical twists were calculated. From the generated fragments it was found that the bond angles are A1=150 degrees +/-2 degrees and A2=130 degrees +/-10 degrees, while the helical twist is 36.6 degrees +/-2 degrees in the A strand and A1=150 degrees +/-6 degrees and A2=130+/-6 degrees with helical twist 39.6 degrees +/-2 degrees in the B strand for the DNA segment with the same sequence as the Dickerson dodecamer.

We describe the development of an in vitro library selection system (CIS display) that exploits the ability of a DNA replication initiator protein (RepA) to bind exclusively to the template DNA from which it has been expressed, a property called cis-activity. A diverse peptide library is created by ligation of DNA fragments of random sequence to a DNA fragment that encodes RepA. After in vitro transcription and translation, a pool of protein-DNA complexes is formed where each protein is stably associated with the DNA that encodes it. These complexes are amenable to the affinity selection of ligands to targets of interest. Here we show that RepA is a highly faithful cis-acting DNA-binding protein and demonstrate that libraries encoding >10(12) random 18-mer peptides can be constructed and used to isolate peptides that bind specifically to disparate targets. The use of DNA to encode the displayed peptides offers advantages over in vitro peptide display systems that use mRNA.