Immune stimulation in the silkworm, Bombyx mori L., by CpG oligodeoxynucleotides

Synthetic ODNs containing unmethylated CpG dinucleotides are known to stimulate immune responses in vertebrates, but so far the effect has not been studied in insects. In this report, we describe an induction of immune response following injection of oligodeoxynucleotides (ODNs) into the insect hemocoel. The fifth instar silkworm (Bombyx mori L.) larvae were injected with several synthetic ODNs containing variable number of unmethylated CpG motifs, heat-denatured genomic DNA of B. mori itself, or intact genomic DNA to observe a new induction pattern in the insect immune mechanism. When the induction of immune response was examined based on the expression rates of genes for antibacterial peptides such as attacin and cecropin, we could confirm that it was triggered upon injection of ODNs. The expression was, however, neither dependent on numbers of CpG motifs nor methylation of CpGs in ODNs. Furthermore, it was confirmed that the presence of CpG in ODN was not involved in the induction pattern of insect immunity caused by ODNs, although it has been reported that vertebrates respond in a specific manner against invading ODNs containing CpG dinucleotides. In addition, insect immunity was not stimulated by injection of intact DNA from host. In contrast, the injection of denatured genomic DNA provoked the host immune reaction. Taken together, our data suggest that foreignness of ODNs or DNA might be a key factor in the induction of insect immunity.     

CpG oligodeoxynucleotides protect normal and SIV-infected macaques from Leishmania infection

Oligodeoxynucleotides containing CpG motifs (CpG ODNs) mimic microbial DNA and activate effectors of the innate immune response, which limits the spread of pathogens and promotes an adaptive immune response. CpG ODNs have been shown to protect mice from infection with intracellular pathogens. Unfortunately, CpG motifs that optimally stimulate humans are only weakly active in mice, mandating the use of nonhuman primates to monitor the activity and safety of "human" CpG ODNs in vivo. This study demonstrates that CpG ODN treatment of rhesus macaques significantly reduces the severity of the lesions caused by a challenge with Leishmania: Leishmania superinfection is common in immunocompromised hosts, particularly those infected with HIV. This study shows that PBMCs from HIV-infected subjects respond to stimulation with CpG ODNs. To determine whether CpG ODNs can protect retrovirus-infected primates, SIV-infected macaques were treated with CpG ODNs and then challenged with Leishmania: Both lesion size and parasite load were significantly reduced in the CpG-treated animals. These findings support the clinical development of CpG ODNs as immunoprotective agents in normal and HIV-infected patients.     

Nucleosides and nucleotides. Part 214: thermal stability of triplexes containing 4'alpha-C-aminoalkyl-2'-deoxynucleosides

In order to develop novel antigene molecules forming thermally stable triplexes with target DNAs and having nuclease resistance properties, we synthesized oligodeoxynucleotides (ODNs) with various lengths of aminoalkyl-linkers at the 4'alpha position of thymidine and the aminoethyl-linker at the 4'alpha position of 2'-deoxy-5-methylcytidine. Thermal stability of triplexes between these ODNs and a DNA duplex was studied by thermal denaturation. The ODNs containing the nucleoside 2 with the aminoethyl-linker or the nucleoside 3 with the aminopropyl-linker thermally stabilized the triplexes, whereas the ODNs containing the nucleoside 1 with the aminomethyl-linker or the nucleoside 4 with the 2-[N-(2-aminoethyl)carbamoyl]oxy]ethyl-linker thermally destabilized the triplexes. The ODNs containing 2 were the most efficient at stabilizing the triplexes with the target DNA. The ODNs containing 4'alpha-C-(2-aminoethyl)-2'-deoxy-5-methylcytidine (5) also efficiently stabilized the triplexes with the target DNA. Stability of the ODN containing 5 to nucleolytic hydrolysis by snake venom phosphodiesterase (a 3'-exonuclease) was studied. It was found that the ODN containing 5 was more resistant to nucleolytic digestion by the enzyme than an unmodified ODN. In a previous paper, we reported that the ODNs containing 2 were more resistant to nucleolytic digestion by DNase I (an endonuclease) than the unmodified ODNs. Thus, it was found that the ODNs containing 4'alpha-C-(2-aminoethyl)-2'-deoxynucleosides were good candidates for antigene molecules.     

Bis-pyrene-labeled oligonucleotides: sequence specificity of excimer and monomer fluorescence changes upon hybridization with DNA

The design, synthesis, and properties of a new pyrene excimer-forming probe of DNA have been described. 2,2-(Aminomethyl)propanediol was converted by the reaction with 1-pyrenebutylic acid to bis-pyrene-modified propanediol as a fluorescent non-nucleosidic linker. The bis-pyrene-modified linker can be incorporated via phosphoramidite chemistry into the 5'-terminal or internal positions of oligonucleotides (ODNs). The terminally modified ODNs showed almost similar affinity for complementary DNA when compared with the corresponding unmodified ODNs. The duplexes containing the bis-pyrene in the main chain exhibited higher melting temperatures relative to the corresponding duplexes containing propanediol linker at the same position. The UV and CD spectral studies indicate that the stacking interactions between the pyrene and DNA bases occur in the internally modified duplex and do not in the terminally modified duplex. The bis-pyrene modified linker itself displays excimer (E at 480 nm) and monomer (M at 380 nm) emission in a quantum yield (QY) of 0.17 and the E/M intensity ratio of 15. Incorporation of this linker into the terminal or internal positions of ODNs reduced the QY (0.003-0.009) and the E/M ratio (0.3-0.8). While small changes in the QY and E/M ratio was obtained in binding of the internally labeled ODNs to DNA, up to 27-fold increase in the QY and 17-fold increase in the E/M ratio was observed upon hybridization of the terminally labeled ODNs with DNA. The excimer and monomer fluorescence changes were found to be sensitive to a mismatch base present in the target DNA. The bis-pyrene-modified ODNs thus provide a sequence-sepcific fluorescent probe of DNA.     

An anti-parallel triple helix motif with oligodeoxynucleotides containing 2'-deoxyguanosine and 7-deaza-2'-deoxyxanthosine.

Triple helix formation of oligodeoxynucleotides (ODNs) with a 15 base pair poly-purine DNA target in the HER2 promoter was examined by footprinting analysis. 7-deaza-2'-deoxyxanthosine (dzaX) was identified as a purine analogue of thymidine (T) which forms dzaX:A-T triplets. ODNs containing 2'-deoxyguanosine (G) and dzaX were found to form triple helices in an anti-parallel orientation, with respect to the poly-purine strand of the target DNA. In comparative studies under physiological K+ and Mg++ concentrations and at pH 7.2, the ODNs containing G and dzaX showed high affinity to the target sequence while the ODNs containing G and T were not able to bind. In the absence of added monovalent salts both ODNs showed high affinity to the target sequence. The substitution of 7-deaza-2'-deoxyguanosine for G substantially decreased the capacity of the ODNs to form triple helices under physiological conditions, indicating that dzaX may be unique in its ability to enhance triple helix formation in the anti-parallel motif.     

G/C-modified oligodeoxynucleotides with selective complementarity: synthesis and hybridization properties.

Modified oligodeoxyribonucleotides (ODNs) that have unique hybridization properties were designed and synthesized for the first time. These ODNs, called selective binding complementary ODNs (SBC ODNs), are unable to form stable hybrids with each other, yet are able to form stable, sequence specific hybrids with complementary unmodified strands of nucleic acid. To make SBC ODNs, deoxyguanosine (dG) and deoxycytidine (dC) were substituted with deoxyinosine (dI) and 3-(2'-deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one (dP), respectively. The hybridization properties of several otherwise identical complementary ODNs containing one or both of these nucleoside analogs were studied by both UV monitored thermal denaturation and non-denaturing PAGE. The data showed that while dI and dP did form base pairs with dC and dG, respectively, dI did not form a stable base pair with dP. A self-complementary ODN uniformly substituted with dI and dP acquired single-stranded character and was able to strand invade the end of a duplex DNA better than an unsubstituted ODN. This observation implies that SBC ODNs should effectively hybridize to hairpins present in single-stranded DNA or RNA.     

Nucleosides and nucleotides. 204. Synthesis of oligodeoxynucleotides containing 6'alpha-[N-(Aminoalkyl)carbamoyloxy]-carbocyclic-thymidines and the thermal stability of the duplexes and their nuclease-resistance properties

To construct the nuclease-resistant oligodeoxynucleotides (ODNs) with natural phosphodiester linkages, we synthesized ODNs that contain 6'alpha-[N-(aminoalkyl)carbamoyloxy]-carbocyclic-thymidines (4, 5, and 6). The stability of these ODNs to nuclease hydrolysis was examined by using snake venom phosphodiesterase (3'-exonuclease) and nuclease S1 (endonuclease). It was found that the ODNs containing 4, 5, or 6 were more resistant to both the enzymes than the unmodified ODN. These nuclease-resistant properties are noteworthy, since they have natural phosphodiester linkages. Next, the thermal stabilities of duplexes consisting of these ODNs and either the complementary DNA or RNA were studied by thermal denaturation. The ODNs that contain 4 were found to enhance the thermal stability of the duplexes with the complementary DNA, while those containing 5 or 6 decreased the thermal stability of the ODN-DNA duplexes. On the other hand, all ODNs that contained 4, 5, or 6 decreased the thermal stability of the ODN-RNA duplexes.     

CpG ODNs对肿瘤细胞端粒酶活性及细胞周期和凋亡的影响

应用TRAP PCR ELISA法检测CpGODNs及E .coliDNA对肿瘤细胞端粒酶活性的影响变化 ,同时用流式细胞仪检测细胞周期的变化及凋亡的产生 ,从基因水平探讨其抗肿瘤机制。实验发现活性形式的CpGODNs可显著降低肿瘤细胞端粒酶活性 ,E .coliDNA的下调作用出现在 48h之后 ,二者均可使G0 /G1期细胞含量增加 ,但均未引起凋亡。结果表明 ,CpGODNs及E .coliDNA在基因水平可通过抑制端粒酶活性达到抗肿瘤目的 ,但不能诱导肿瘤细胞凋亡。     

High affinity interaction of HIV-1 integrase with specific and non-specific single-stranded short oligonucleotides.

Retroviral integrase (IN) catalyzes the integration of double-stranded viral DNA into the host cell genome. The reaction can be divided in two steps: 3'-end processing and DNA strand transfer. Here we studied the effect of short oligonucleotides (ODNs) on human immunodeficiency virus type 1 (HIV-1) IN. ODNs were either specific, with sequences representing the extreme termini of the viral long terminal repeats, or nonspecific. All ODNs were found to competitively inhibit the processing reaction with Ki values in the nM range for the best inhibitors. Our studies on the interaction of IN with ODNs also showed that: (i) besides the 3'-terminal GT, the interaction of IN with the remaining nucleotides of the 21-mer specific sequence was also important for an effective interaction of the enzyme with the substrate; (ii) in the presence of specific ODNs the activity of the enzyme was enhanced, a result which suggests an ODN-induced conformational change of HIV-1 IN.     

A label-free optical sensor based on nanoporous gold arrays for the detection of oligodeoxynucleotides

Interest in using nanoporous materials for sensing applications has increased. The present study reports a method of preparing well-ordered nanoporous gold arrays using a porous silicon (PSi) template. Gold nanolayer could be electrodeposited on the surface of the PSi template at low electrolysis currents in low concentration of chloroauric acid (HAuCl₄) solution. Surface morphology characterizations and optical measurements revealed that a PSi-templated nanoporous gold (Au–PSi) array well replicated the nanoporous structure and retained the optical properties of PSi. Fourier transform reflectometric interference spectra showed that a characteristic blue-shifted effective optical thickness (EOT) was observed due to the low refractive index of the gold film. An optical DNA biosensor was then fabricated via the self-assembly of single-stranded DNA (ssDNA) with a specific sequence on the surface of Au–PSi. The attachment of ssDNA and its hybridization with target oligonucleotides (ODNs) persistently caused the blue shift of the EOT. Consequently, a relationship between the EOT shift and the ODN concentration was established. The mechanism of the optical response caused by DNA hybridization on the Au–PSi surface was qualitatively explained by the electromagnetic theory and electrochemical impedance spectroscopy (EIS). The lowest detection limit for target ODNs was estimated at around 10⁻¹⁴ mol L⁻¹, when the baseline noise, a variation in the value of EOT is around 5 nm. The fabricated Au–PSi based optical biosensor has potential use in the discovery of new ODN drugs because it will be able to detect the binding event between ODNs and the target DNA.     

Use of locked nucleic acid oligonucleotides to add functionality to plasmid DNA

The available reagents for the attachment of functional moieties to plasmid DNA are limiting. Most reagents bind plasmid DNA in a non-sequence- specific manner, with undefined stoichiometry, and affect DNA charge and delivery properties or involve chemical modifications that abolish gene expression. The design and ability of oligonucleotides (ODNs) containing locked nucleic acids (LNAs) to bind supercoiled, double-stranded plasmid DNA in a sequence-specific manner are described for the first time. The main mechanism for LNA ODNs binding plasmid DNA is demonstrated to be by strand displacement. LNA ODNs are more stably bound to plasmid DNA than similar peptide nucleic acid (PNA) ‘clamps’ for procedures such as particle-mediated DNA delivery (gene gun). It is shown that LNA ODNs remain associated with plasmid DNA after cationic lipid-mediated transfection into mammalian cells. LNA ODNs can bind to DNA in a sequence-specific manner so that binding does not interfere with plasmid conformation or gene expression. Attachment of CpG-based immune adjuvants to plasmid by ‘hybrid’ phosphorothioate–LNA ODNs induces tumour necrosis factor-α production in the macrophage cell line RAW264.7. This observation exemplifies an important new, controllable methodology for adding functionality to plasmids for gene delivery and DNA vaccination.     

Modulation of DNA Triplex Stability Through Nucleobase Modifications

Abstract

Spermine conjugation at⁴ N of 5-Me-dC in oligonucleotides (sp-ODNs) reduces the net negative charge and these as HG strands form triplexes with foremost stability at neutral pH (7.3), in contrast to unmodified ODNs which form stable triplexes at pH 5.5. The stability of sp-ODN triplexes is shown to arise kom improved association with duplex caused by electrostatic interaction of polycationic spermine sidechain with anionic phosphate backbone of DNA and N3 protonation is not a pre-requirement for triplexes constituted from sp- ODNs. The amplification of electrostatic component of interaction can be achieved by transformation of primary amino group of polyamines to corresponding guanidinium functions leading to improved binding and stabilization of DNA triplexes even at pH 7.0. %-Amino-dU ODNs are shown to be compatible as a central strand in formation of triplexes in which pyrimidine would be in the middle position of a triad.     

Nucleosides and nucleotides. 208. Alternate-strand triple-helix formation by the 3'-3'-linked oligodeoxynucleotides with the anthraquinonyl group at the junction point.

The synthesis of 3'-3'-linked oligodeoxynucleotides (ODNs) with the anthraquinonyl group at the junction point is described. The ODNs were synthesized on a DNA synthesizer using a controlled pore glass (CPG) carrying pentaerythritol that has an intercalator at one of the four hydroxymethyl groups. Stability of the triplexes with the target duplexes was studied by thermal denaturation. The 3'-3'-linked ODNs with the anthraquinonyl group enhanced the thermal stability of the triplexes when compared with those without the intercalator and the unmodified nonamer 10. It was found that the ODNs 12 and 13 carrying the anthraquinonyl groups can form thermally stable triplexes by skipping two or three extra base pairs between two binding domains of the target duplexes. The ability of the 3'-3'-linked ODNs to inhibit cleavage of the target DNA 22 by the restriction enzyme Hind III was tested. It was found that the 3'-3'-linked ODN 16 with the anthraquinonyl group at the junction point inhibited the cleavage by the enzyme more effectively than the nonamer 14 and the 3'-3'-linked ODN 15 without the intercalator.     

内皮素受体反义寡聚核苷酸对内皮素受体基因表达及血管平滑肌细胞增殖的影响

报道了内皮素Ａ型受体反义寡聚核苷酸（ＯＤＮｓ）对大鼠血管平滑肌细胞（ＶＳＭＣ）增殖及内皮素受体基因表达的影响．~３Ｈ－ＴｄＲ参入结果显示,内皮素Ａ型受体反义ＯＤＮｓ处理细胞可显著抑制内皮素诱导的ＶＳＭＣ的ＤＮＡ合成,反转录－ＰＣＲ及受体结合实验结果表明,ＯＤＮｓ的上述作用与降低ＶＳＭＣ内皮素Ａ型受体基因表达活性有关．     

Chemical ligation of oligodeoxynucleotides by X-irradiation and its application to regulation of G-quadruplex formation

We demonstrated radiolytic ligation of oligodeoxynucleotides (ODNs) possessing disulfide bond and its application to regulation of DNA quadruplex formation. G-rich hexamer ODNs had poor ability to form quadruplex, while X-irradiation of the ODNs induced interstrand exchange reaction at disulfide bond to form ligated 12 mer ODNs, leading to the ready formation of quadruplex due to the entropic effect. Since complexation of the ligated ODNs with hemin in the presence of K⁺ showed strong soret band absorption and also catalyzed the H₂O₂-mediated oxidation of luminol, it appears that the quadruplex formed from ligated ODNs showed a function similar to native DNA quadruplex.     

Automated synthesis of new ferrocenyl-modified oligonucleotides: study of their properties in solution

We have developed new ferrocenyl-modified oligonucleotide (ODN) probes for electrochemical DNA sensors. A monofunctional ferrocene containing phosphoramidite group has been prepared, and a new bisfunctional ferrocene containing phosphoramidite and dimethoxytrityl (DMT) groups has been developed. These ferrocenyl-phosphoramidites have been directly employed in an automated solid-phase DNA synthesizer using phosphoramidite chemistry. The advantages of this method are that it allows a non-specialist in nucleotide chemistry to access labeled ODNs and that it has demonstrated good results. ODNs modified at the 3′ and/or 5′ extremities have been prepared, with the incorporation of the ferrocenyl group into the chain. The 5′ position appears to be more important due to its particular behavior. The thermal stability and electrochemical properties of these new ODN ferrocenes were analyzed before and after hybridization with different ODNs. The feasibility of using these new ferrocenyl-labeled ODNs in DNA sensors has been demonstrated.     

Preparation of oligodeoxynucleotides containing 5-(N-methylpiperazinyl) and 5-benzyloxymethyl uracils

Deprotected compounds 1 and 9 were allowed to react with 4,4'-dimethoxytrityl chloride in pyridine to give 5'-O-DMT nucleosides 2 and 10. The 3'-phosphoramidites 4 and 11 were incorporated into oligodeoxynucleosides (ODNs). The hybridization properties of the modified ODNs with their complementary DNA strands were studied. Interesting results were obtained when 11 was inserted as a bulged nucleoside into TWAs, duplexes, and triplexes.     

3′-Modified oligodeoxyribonucleotides for the study of 2-deoxyribose damage in DNA

Well-defined substrates for the study of oxidative processes are important for the elucidation of the role of DNA damage in the etiology of diseases such as cancer. We have synthesized 3′-modified oligodeoxyribonucleotides (ODNs) using 5′ → 3′ ‘reverse’ DNA synthesis for the study of 2-deoxyribose oxidative damage to DNA. The modified monomers designed for these studies all share a common feature, they lack the naturally occurring 3′-hydroxyl group found in 2-deoxyribonucleosides. Modified H-phosphonates containing 3′-phenyl selenides as well as saturated and unsaturated sugars were obtained and incorporated in ODNs. These ODNs were used to investigate the fate of C3′-dideoxyribonucleotide radicals in DNA.     

Reduced aggregation and improved specificity of G-rich oligodeoxyribonucleotides containing pyrazolo[3,4-d]pyrimidine guanine bases

Guanine (G)-rich oligodeoxyribonucleotides (ODNs) can form undesired complexes by self association through non-Watson–Crick interactions. These aggregates can compromise performance of DNA probes and make genetic analysis unpredictable. We found that the 8-aza-7-deazaguanine (PPG), a pyrazolo[3,4-d]pyrimidine analog, reduces guanine self association of G-rich ODNs. In the PPG heterocycle, the N-7 and C-8 atoms of G are interposed. This leaves the ring system with an electron density similar to G, but prevents Hoogsteen-bonding associated with N-7. ODNs containing multiple PPG bases were easily prepared using a dimethylformamidine-protected phosphoramidite reagent. Substitution of PPG for G in ODNs allowed formation of more stable DNA duplexes. When one or more PPGs were substituted for G in ODNs containing four or more consecutive Gs, G aggregation was eliminated. Substitution of PPG for G also improved discrimination of G/A, G/G and G/T mismatches in Watson–Crick hybrids. Use of PPG in fluorogenic minor groove binder probes was also explored. PPG prevented aggregation in MGB probes (MGB^TM is a trademark of Epoch Biosciences) and allowed use of G-rich sequences. An increased signal was observed in 5′-PPG probes due to reduced quenching of fluorescein by PPG. In summary, substitution of PPG for G enhances affinity, specificity, sensitivity and predictability of G-rich DNA probes.