Oligodeoxynucleotides containing C-7 propyne analogs of 7-deaza-2'-deoxyguanosine and 7-deaza-2'-deoxyadenosine.

The synthesis, hybridization properties and antisense activities of oligodeoxynucleotides (ODNs) containing 7-(1-propynyl)-7-deaza-2'-deoxyguanosine (pdG) and 7-(1-propynyl)-7-deaza-2'-deoxyadenosine (pdA) are described. The suitably protected nucleosides were synthesized and incorporated into ODNs. Thermal denaturation (Tm) of these ODNs hybridized to RNA demonstrates an increased stability relative to 7-unsubstituted deazapurine and unmodified ODN controls. Antisense inhibition by these ODNs was determined in a controlled microinjection assay and the results demonstrate that an ODN containing pdG is approximately 6 times more active than the unmodified ODN. 7-Propyne-7-deaza-2'-deoxyguanosine is a promising lead analog for the development of antisense ODNs with increased potency.     

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.     

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.     

PyNTTTTGT and CpG Immunostimulatory Oligonucleotides: Effect on Granulocyte/Monocyte Colony-Stimulating Factor (GM-CSF) Secretion by Human CD56+ (NK and NKT) Cells

CD56⁺ cells have been recognized as being involved in bridging the innate and acquired immune systems. Herein, we assessed the effect of two major classes of immunostimulatory oligonucleotides (ODNs), PyNTTTTGT and CpG, on CD56⁺ cells. Incubation of human peripheral blood mononuclear cells (hPBMC) with some of these ODNs led to secretion of significant amounts of interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α) and granulocyte/monocyte colony-stimulating factor (GM-CSF), but only if interleukin 2 (IL2) was present. IMT504, the prototype of the PyNTTTTGT ODN class, was the most active. GM-CSF secretion was very efficient when non-CpG ODNs with high T content and PyNTTTTGT motifs lacking CpGs were used. On the other hand, CpG ODNs and IFNα inhibited this GM-CSF secretion. Selective cell type removal from hPBMC indicated that CD56⁺ cells were responsible for GM-CSF secretion and that plasmacytoid dendritic cells (PDCs) regulate this process. In addition, PyNTTTTGT ODNs inhibited the IFNα secretion induced by CpG ODNs in PDCs by interference with the TLR9 signaling pathway. Since IFNα is essential for CD56⁺ stimulation by CpG ODNs, there is a reciprocal interference of CpG and PyNTTTTGT ODNs when acting on this cell population. This suggests that these synthetic ODNs mimic different natural alarm signals for activation of the immune system.     

Effects of 5-(N-aminohexyl)carbamoyl-2'-deoxyuridine on endonuclease stability and the ability of oligodeoxynucleotide to activate RNase H.

To evaluate an endonuclease resistance property of oligodeoxynucleotides (ODNs) containing 5-(N-aminohexyl)carbamoyl-2'-deoxyuridines (Hs) and to elucidate whether a duplex consisting of the ODN analogue and its complementary RNA induces RNase H activity, the ODNs containing the deoxyuridine analogues, Hs, at intervals of one, two, three, four and five natural nucleosides were synthesized. From partial hydrolysis of these ODNs with nuclease S1 (an endonuclease), it was found that the ODNs became more stable towards nucleolytic hydrolysis by the enzyme as the number of H increased. Furthermore, to examine whether the duplexes composed of the ODNs containing Hs and their complementary RNAs are substrates for RNase H or not, the duplexes of these ODNs and their complementary RNA strands were treated with Escherichia coliRNase H. It was found that cleavage of the RNA strands by the enzyme was kinetically affected by the introduction of Hs into the duplexes.     

Intracellular traffic of oligodeoxynucleotides in and out of the nucleus: effect of exportins and DNA structure

The delivery of oligodeoxynucleotides (ODNs) into cells is widely utilized for antisense, antigene, aptamer, and similar approaches to regulate gene and protein activities based upon the ODNs' sequence-specific recognition. Short pieces of DNA can also be generated in biological processes, for example, after degradation of viral or bacterial DNA. However, the mechanisms that regulate intracellular trafficking and localization of ODNs are not fully understood. Here we study the effects of major transporters of microRNA, exportin-1 (Exp1) and exportin-5 (Exp5), on the transport of single-stranded ODNs in and out of the nucleus. For this, we employed a fluorescent microscopy-based assay to quantitatively measure the redistribution of ODNs between the nucleus and cytoplasm of live cells. By measuring the fluorescent signal of the nuclei we observed that after delivery into cells via cationic liposomes ODNs rapidly accumulated inside nuclei. However, after removal of the ODN/liposome containing media, we found re-localization of ODNs from the nuclei to cytoplasm of the cells over the time course of several hours. Downregulation of the Exp5 gene by siRNA resulted in a slight increase of ODN uptake into the nucleus, but the kinetics of ODN efflux to the cytoplasm was not affected. Inhibition of Exp1 with leptomycin B somewhat slowed down the clearance of ODNs from the nucleus; however, within 6 hours most of the ODN were still being cleared form the nucleus. ODNs that could form intramolecular G-quadruplex structures behaved differently. They also accumulated in nuclei, although at a lesser extent than unstructured ODN, but they remained there for up to 20 hours after transfection, causing significant cell death. We conclude that Exp1 and Exp5 are not the major transporters of our ODNs out of the nucleus, and that the transport of ODNs is strongly affected by their secondary structure.     

Detection of CAG repeat DNA sequences by pyrene-functionalized pyrrole-imidazole polyamides

Five N-methylpyrrole-N-methylimidazole (Py-Im) polyamides possessing a fluorescent pyrene were synthesized by Fmoc solid-phase synthesis using Py/Im monomers and pyrenylbutyl-pyrrole monomer compound 9. The steady state fluorescence of conjugates 1-5 was examined in the presence and absence of (CAG)(12)-containing oligodeoxynucleotides (ODNs) 1 and 2. Of the conjugates, conjugate 1 showed no background emission around 470 nm in the absence of ODNs, and a clear increase of emission at 475 nm was observed upon addition of ODNs 1 and 2. The emission of conjugate 1 at 475 nm increased linearly with the concentration of ODN and the number of CAG repeats. The results indicate that conjugate 1 efficiently forms a pyrene excimer upon binding in the minor groove of DNA.     

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.     

Antisense hairpin loop oligonucleotides as inhibitors of expression of multidrug resistance-associated protein 1: their stability in fetal calf serum and human plasma

Multidrug resistance-associated protein (MRP1) is a transmembrane pump protein responsible for the efflux of chemotherapeutic drugs, an important cause of anticancer treatment failure. Trying to circumvent MRP-mediated resistance we designed and synthesized hairpin loops forming antisense oligodeoxyribonucleotides (ODNs), both phosphodiesters (PO-ODNs) and their phosphorothioate analogues (PS-ODNs), to reduce the protein expression by targeting its mRNA in a sequence specific manner. Melting temperature measurements as well as polyacrylamide gel electrophoresis supported the preferential formation of a secondary structure, which was expected to protect ODNs against 3'-exonuclease degradation. ODNs and PS-ODNs designed in this work were successfully tested as antisense inhibitors of the expression of MRP1 in the leukaemia HL60/ADR cell line. Foreseeing the necessity to perform clinical studies with such ODNs we investigated their stability against the 3'-exonuclease activity of fetal calf serum and human plasma. Under the conditions, corresponding to physiological ones, we observed high stability of hairpin loop forming ODNs, especially those containing longer (e.g. 7 base pair) stems. Comparative studies on the stability of chemically unmodified hairpin loop forming ODNs and their PS-counterparts indicated that endonuclease activity did not play any important role in the process of their nucleolytic degradation. Our studies provide strong evidence for high stability of chemically unmodified hairpin loop ODNs, making them an attractive alternative to phosphorothioate analogues commonly used in antisense strategy.     

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．     

Oxetane modified, conformationally constrained, antisense oligodeoxyribonucleotides function efficiently as gene silencing molecules

Incorporation of nucleosides with novel base-constraining oxetane (OXE) modifications [oxetane, 1-(1',3'-O-anhydro-beta-d-psicofuranosyl nucleosides)] into antisense (AS) oligodeoxyribonucleotides (ODNs) should greatly improve the gene silencing efficiency of these molecules. This is because OXE modified bases provide nuclease protection to the natural backbone ODNs, can impart T(m) values similar to those predicted for RNA-RNA hybrids, and not only permit but also accelerate RNase H mediated catalytic activity. We tested this assumption in living cells by directly comparing the ability of OXE and phosphorothioate (PS) ODNs to target c-myb gene expression. The ODNs were targeted to two different sites within the c-myb mRNA. One site was chosen arbitrarily. The other was a 'rational' choice based on predicted hybridization accessibility after physical mapping with self-quenching reporter molecules (SQRM). The Myb mRNA and protein levels were equally diminished by OXE and PS ODNs, but the latter were delivered to cells with approximately six times greater efficiency, suggesting that OXE modified ODNs were more potent on a molar basis. The rationally targeted molecules demonstrated greater silencing efficiency than those directed to an arbitrarily chosen mRNA sequence. We conclude that rationally targeted, OXE modified ODNs, can function efficiently as gene silencing agents, and hypothesize that they will prove useful for therapeutic purposes.     

Oligodeoxynucleotides having a loop consisting of 3'-deoxy-4'-C-(2-hydroxyethyl)thymidines form stable hairpins

Components that form stable hairpin loops are highly useful for the development of functional DNA and RNA molecules. We have designed and synthesized a sugar-modified thymidine analogue, 3'-deoxy-4'-C-(2-hydroxyethyl)thymidine (X), as a nucleosidic loop component stabilizing the hairpin structure. The ODNs I-1-4, 5'-d[CGAACG-X(n)-CGTTCG]-3' (I-1, n = 1; I-2, n = 2; I-3, n = 3; I-4, n = 4), forming the hairpin loop structures, of which the loop moiety consisted of the analogue X, and also the corresponding unmodified ODNs II-1-4, 5'-d[CGAACG-T(n)-CGTTCG]-3' (II-1, n = 1; II-2, n = 2;II-3, n = 3; II-4, n = 4), having a thymidine loop, were synthesized by the phosphoramidite method. The melting temperatures (T(m)) of the ODNs I-1-4 containing X in the loop moiety at 5 microM were 67.1, 68.1, 73.0, and 69.3 degrees C, respectively, and those of the control natural ODNs II-1-4 were 65.3, 67.0, 69.2, and 68.8 degrees C, respectively. Thus, the ODNs I-1-4 formed a more thermally stable hairpin than the corresponding unmodified ODNs II-1-4 having an equal number of loop residues. The hairpin structures of the modified ODNs I-1-4 and the unmodified ODNs II-1-4 were investigated by CD spectroscopy and molecular mechanics calculations. These results showed that the 4'-branched nucleoside X can stabilize hairpin structures when it is present in the loop moiety, probably due to the flexibility of the one-carbon-elongated 4'-branched structure.     

Synthesis of oligodeoxyribonucleotides bearing a functional group at 2'-position by post-synthetic modification.

A novel 2'-thio-2'-deoxyuridine derivative bearing a reactive ester at 2'-position was synthesized and incorporated into oligodeoxyribonucleotides (ODNs) for the post-synthetic modification of ODNs. This ODNs was reacted with several amines to give ODNs having a functional group at 2'-position.     

DNA-enzyme-mediated cleavage of human immunodeficiency virus type 1 Gag RNA is significantly augmented by antisense-DNA molecules targeted to hybridize close to the cleavage site

DNA-enzymes (Dzs) usually cleave short synthetic target RNAs very efficiently, but this activity diminishes significantly when tested on full-length RNAs, primarily because of the rigid secondary structures near the target sequence. We identified two Dzs, one each for 81-17 and 10-23 Dz, which cleaved the human immunodeficiency virus type 1 (HIV-1) Gag RNA poorly. We sought to use short oligodeoxynucleotides (ODNs) with the hope that it will facilitate Dz-mediated cleavage. The efficiencies of several ODNs were analyzed for their ability to augment the 8-17 Dz-mediated cleavage. We observed that ODNs that hybridized close to 5' and 3' ends of the target sequence were able to enhance significantly 8-17 Dz-mediated cleavage activity in a dose-dependent manner. The same was true for 10-23 Dz with ODNs that hybridized close to the target site. Thus, it was possible to enhance significantly the cleavage activity of poorly cleaving HIV-1 Gag-specific Dzs by using sequence-specific ODNs. This combination of antisense and catalytic Dz will, in principle, result in more effective gene suppression that could be exploited for therapeutic purposes.     

The application of antisense oligonucleotide technology to the brain: Some pitfalls

Summary 1. Amphetamine-induced c-fos andegr-1 expression in the striatum was used as a model in which to study the effects of antisense oligodeoxynucleotides (ODNs) directed at c-fos. Using direct infusions of ODNs into the striata of animals we have demonstrated that c-fos antisense ODNs retain most of their biological activity with 2- or 3-base substitutions. The c-fos antisense and mismatch ODNs attenuated Fos immunoreactivity but had little effect on Egr-1 immunoreactivity.2. In another group of studies examining the role of c-fos in amygdala kindling, we have demonstrated that ODNs cause neurotoxic damage following repeated daily infusions into the amygdala. The damage observed was greatly diminished when the time interval between infusions was extended.     

Necessity of oligonucleotide aggregation for toll-like receptor 9 activation

Toll-like receptor 9 (TLR9), a member of the interleukin-1 (IL-1) family of pathogen-associated molecular pattern receptors, is activated by unmethylated CpG-containing sequences in bacterial DNA or synthetic oligonucleotides (ODNs) in the endosomal compartment. The stimulation of an IL-1 response is thought to require the aggregation of its receptor. By analogy, we postulated that the potency of a TLR9 ligand should depend first on its ability to enter cells and gain access to TLR9 and second on its capacity to form a multimeric complex capable of cross-linking these receptors. Previously, we selected from a random library a series of phosphodiester ODNs with enhanced ability to permeate cells. Here, we studied the structural requirements for these penetrating ODNs to elicit a functional TLR9 response, as assessed by cytokine production from bone marrow-derived mouse mononuclear cells. The presence of a prototypic murine immunostimulatory DNA hexameric sequence (purine-purine-CG-pyrimidine-pyrimidine) in the ODNs was not sufficient for stimulation. In addition, the TLR9-activating ODNs had to have the ability to form aggregates and often to form secondary structures near the core CpG motifs. Multimerization was promoted by the presence of a guanine-rich 3'-terminus. The phosphodiester ODNs with CpG motifs that did not aggregate antagonized the effects of the multimeric TLR9 activators. These findings suggest that an optimal TLR9 agonist needs to contain a spatially distinct multimerization domain and a receptor binding CpG domain. This concept may prove useful for the design of new TLR9-modulating agents.