Oligonucleotides最新文献

Anti-human immunodeficiency virus-1 (HIV-1) polyamide (peptide) nucleic acids (PNAs) conjugated with cell-penetrating peptides (CPPs) targeted to the viral genome are potent virucidal and antiviral agents. Earlier, we have shown that the anti-HIV-1 PNA(TAR)-penetratin conjugate is rapidly taken up by cells and is nontoxic to mice when administered at repeat doses of as high as 100 mg/kg body weight. In the present studies we demonstrate that naked PNA(TAR) is immunologically inert as judged by the proliferation responses of splenocytes and lymph node cells from PNA(TAR)-immunized mice challenged with the immunizing antigen. In contrast, PNA(TAR)-penetratin conjugate is moderately immunogenic mainly due to its penetratin peptide component. Cytokine secretion profiles of the lymph node cells from the conjugate-immunized mice showed marginally elevated levels of proinflammatory cytokines, which are known to promote proliferation of T lymphocytes. Since the candidate compound, PNA(TAR)-penetratin conjugate displays potent virucidal and antiviral activities against HIV-1, the favorable immunological response together with negligible toxicity suggest a strong therapeutic potential for this class of compounds.

RNA interference offers enormous potential to develop therapeutic agents for a variety of diseases. To assess the stability of siRNAs under conditions relevant to clinical use with particular emphasis on topical delivery considerations, a study of three different unmodified siRNAs was performed. The results indicate that neither repeated freeze/thaw cycles, extended incubations (over 1 year at 21 degrees C), nor shorter incubations at high temperatures (up to 95 degrees C) have any effect on siRNA integrity as measured by nondenaturing polyacrylamide gel electrophoresis and functional activity assays. Degradation was also not observed following exposure to hair or skin at 37 degrees C. However, incubation in fetal bovine or human sera at 37 degrees C led to degradation and loss of activity. Therefore, siRNA in the bloodstream is likely inactivated, thereby limiting systemic exposure. Interestingly, partial degradation (observed by gel electrophoresis) did not always correlate with loss of activity, suggesting that partially degraded siRNAs retain full functional activity. To demonstrate the functional activity of unmodified siRNA, EGFP-specific inhibitors were injected into footpads and shown to inhibit preexisting EGFP expression in a transgenic reporter mouse model. Taken together, these data indicate that unmodified siRNAs are viable therapeutic candidates.

Selection of optimal antisense constructs is still a problem. Among a huge number of antisense oligonucleotides (AS-ONs) only a small piece show inhibitory efficacy. We want to develop an enhanced strategy for specific selection of effective AS-ONs based on prediction of secondary structure of the target messenger RNA (mRNA) and analysis of thermodynamic properties of the mRNA/AS-ON hybrid. Numerous AS-ONs targeted on human tissue factor (TF) mRNA were investigated to evaluate the relevance of different thermodynamic and structural properties on inhibitory efficacy. Cell viability, TF protein and TF mRNA were determined after transfection of bladder cancer cell line J82. Inhibitory efficacy was related to GC content, target region within the TF mRNA and stability of the mRNA/AS-ON hybrid or affinity of the AS-ON to the target mRNA. We found effective AS-ONs targeted on translated region or 3'-untranslated region of TF RNA. We also detected a great correlation between inhibitory efficacy and GC content as well as stability of the mRNA/AS-ON hybrid.

Two types of reporters for optical sensing of NF-kappaB p50 protein-oligodeoxyribonucleotide (ODN) duplex interactions were designed and compared in vitro. The reporters were based on the effect of fluorescence resonance energy transfer (FRET) between the pair donor Cy5.5 near-infrared (NIR) fluorochrome and either 800CW emitting fluorescence dye acceptor (800CW-Cy), or a nonemitting QSY 21 dye quencher (QSY-Cy). The donor and the acceptor dyes were covalently linked to the complementary oligonucleotides, respectively: Cy dye was conjugated to 3'-thiol, whereas 800CW or QSY21 were conjugated to a hydrophilic internucleoside phosphate amino linker. The reporters were tested initially using recombinant NF-kappaB p50 protein binding assays. Both reporters were binding p50 protein, which protected oligonucleotide duplex from degradation in the presence of exonuclease.The incubation of 800CW-Cy reporter in the presence of control or IL-1beta treated human endothelial cells showed the uptake of the reporter in the cytoplasm and the nucleus. The measurement of NIR fluorescence ratio (i.e. Cy5.5/800CW) showed a partial loss of FRET and the increased Cy5.5 fluorescence in nontreated, control cells. Thus, the specific p50 binding to ODN duplex reporters affected the donor-acceptor fluorochrome pair. NF-kappaB p50 exhibited the protective effect on FRET between NIR fluorochromes linked to the complementary strands of the reporter duplex.

We have previously shown that Dz13, a catalytic DNA molecule (DNAzyme) designed against c-jun, is cytotoxic to nonquiescent cells by a mechanism independent of c-jun mRNA cleavage. In this report, we evaluated programmed cell death (PCD) pathways in order to gain further insight into the mechanism of action of Dz13. Using human dermal microvascular endothelial cells (HMEC-1), we found that Dz13-mediated cell death is characterized by mitochondrial depolarization, caspase-8 activation, lysosomal increase, and autophagosome formation. Classical DNA laddering and translocation of mitochondrial proteins were not observed. An array of inhibitors, including those targeting caspases, failed to abrogate cytotoxicity and mitochondrial depolarization. Cytotoxicity did not proceed from endoplasmic reticulum (ER) stress. The possible involvement of PARP-1 in Dz13-mediated cytotoxicity was indicated by its differential release as gauged by protein extraction data and its apparent binding to Dz13, as evidenced by protein pull-down experiments. This study on Dz13-mediated cytotoxicity presents a detailed investigation into the interplay of cell death effectors involved in apoptosis, autophagy, and necrosis, and demonstrates a novel form of oligonucleotide-mediated cytotoxicity with features of PCD.

A multitarget approach is needed for effective gene silencing that combines more than one antiviral strategy. With this in mind, we designed a wild-type (wt) and selectively disabled chimeric mutant (mt) constructs that consisted of small hairpin siRNA joined by a short intracellular cleavable linker to a known hammerhead ribozyme, both targeted against the full-length X RNA of hepatitis B. These chimeric RNAs possessed the ability to cleave the target RNA under in vitro conditions and were efficiently processed at the cleavable site. When this wt chimeric RNA construct was introduced into a liver-specific mammalian cell line, HepG2, along with the HBx substrate encoding DNA, very significant (approximately 70%) intracellular downregulation in the levels of target RNA was observed. When the siRNA portion of this chimeric construct was mutated, keeping the ribozyme (Rz) region unchanged, it caused only approximately 25% intracellular reduction. On the contrary, when only the Rz was made catalytically inactive, about 55% reduction in the target RNA was observed. Construct possessing mt Rz and mt siRNA caused only 10% reduction. This wt chimeric construct also resulted in almost complete knockdown of intracellular HBx protein production, and the mt versions were less effective. The intracellular reduction of target RNA with either wt or mt constructs also interfered with the known functions of HBx protein with varying efficiencies. Thus, in this proof of concept study we show that the levels of the target RNA were reduced potently by the wt chimeric siRNA-Rz construct, which could be modulated with mt versions of the same.

Molecular imaging was used to study the biodistribution, pharmacokinetics, and activity of naked small interfering RNAs (siRNAs). siRNAs with riboses chemically modified in the 2' position were compared with unmodified siRNA. In vitro, replacement of the 2'-hydroxyl (2'OH) group of certain nucleotides in an siRNA sequence by a fluorine atom (2'F) on both antisense (AS) and sense (S) strands [2'F(AS/S)], or by a methoxy group (2'OMe) on the S strand [2'OH(AS)/2'OMe(S)], was compatible with RNA interference. Different siRNAs [2'F(AS/S), 2'OH(AS)/2'OMe(S), and 2'OH(AS/S)] were labeled with fluorine-18 (conjugation with [(18)F]FPyBrA), and comparative dynamic and quantitative imaging was performed with positron emission tomography. After intravenous injections of [(18)F]siRNAs in rodents, total radioactivity was rapidly eliminated by the kidneys and the liver. Tissue distribution of the different siRNAs were similar, and their bioavailability (as judged from blood persistence and stability) increased in the order 2'OH(AS/S) = 2'OH(AS)/2'OMe(S) < 2'F(AS/S). However, in our in vivo model, the 2'F(AS/S) siRNA, despite its higher bioavailability, was not able to induce a higher interference effect with respect to the 2'OH(AS/S) siRNA. Molecular imaging approaches, applied in the present work to both natural and chemically modified siRNAs, can contribute to the development of these macromolecules as therapeutic agents.

A simple and sensitive method for electrochemical detection of DNA was designed. This DNA sensor was based on a "sandwich" detection strategy, which involved a long capture probe DNA immobilized on glassy carbon electrodes that flanked both the reference DNA and target DNA. Electrochemical signals were measured by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) using aquadichloro(benzimidazole)-copper(II), Cu(bzim)(H(2)O)Cl(2), as an electroactive indicator. An improving amount of Cu(bzim)(H(2)O)Cl(2) was interacted with the hybrid DNA via the incorporation of a long-probe DNA and a reference DNA in this sensor. As a result of this effect, this sensor design significantly enhanced the sensitivity. With 48-mer probe DNA and 27-mer reference DNA, the proposed method could be used for detection of 21-mer ssDNA ranging from 1.32 x 10(-7) to 2.52 x 10(-6) M with a detection limit of 2.94 x 10(-8) M. Electrochemical DNA biosensors were also developed using the same long-probe sequence as the target sequence with the novel hybridization indicator, Cu(bzim) (H(2)O)Cl(2). The detection limits for the complementary 21-mer target and 27-mer target were 9.52 x 10(-8) M and 5.81 x 10(-8) M, respectively. The results showed that the sensor with long-probe DNA and reference DNA is far more sensitive than that with nonswitch assay.

10-23 DNAzyme is an oligodeoxyribonucleotide-based ribonuclease. It consists of a 15-nt catalytic domain flanked by two target-specific complementary arms. It has been shown to cleave target mRNA effectively at purine (R)-pyrimidine (Y) dinucleotide. Taking advantage of this specific property, 10-23 DNAzyme was designed to cleave mRNA of a given allele at a unique RY dinucleotide while leaving the mRNA encoded from other alleles of the same gene intact. In this study, a p53-R249S (AGG-->AGT) mutant was tested. 10-23 DNAzyme was used to cut mutant mRNA at GT dinucleotide of codon 249. Both in vitro and in vivo studies showed that this DNAzyme could specifically cut the mutant p53 allele, leaving the wild-type unaffected. This proof-of-concept experiment provided a new way to knock down expression of a given allele with special single-base transversion.