Oligonucleotides最新文献

Oligonucleotides (ONs) such as antisense oligonucleotides (AS-ON) and siRNAs are used as experimental tools to study gene function and are currently being tested in clinical trials for use as therapeutic anticancer agents. However, their therapeutic use has been limited by their low physiological stability and their slow cellular uptake. The systemic delivery of sequence-specific AS-ON targeting the EWS/FLI1 gene product by a targeted, nonviral delivery system dramatically inhibits tumor growth in a murine model of Ewing's sarcoma. The nonviral delivery system uses a poly-iso-hexyl-cyanoacrylate (PIHCA)-containing polycation (chitosan) to bind and protect the AS-ON. No antitumor effect is observed using a control oligonucleotide sequence. We found here that injection of the free AS-ON stimulates tumor growth independently of its sequence and that this stimulation is abolished in the presence of nanosphere-chitosan, which exerts with the oligonucleotides a specific inhibitory effect on tumor growth. The stimulation of tumor growth is likely to be due to a polyanionic effect; indeed, a similar stimulatory response is observed upon treatment with dextran sulfate and heparin in vivo. These results suggest that ON loaded onto nanosphere-chitosan provides efficient and tumor-specific delivery, and provides protection against a polyanionic secondary effect.

Myasthenia gravis (MG) is mainly caused by autoantibodies to postsynaptic nicotinic acetylcholine receptors (AChRs). Previously, we isolated an RNA aptamer with 2'-amino pyrimidines that inhibited both a rat monoclonal antibody, which recognizes the main immunogenic region on the AChR, and MG patient autoantibodies from down-modulating AChRs on human cells. In this study, secondary configuration and binding motif of the aptamer were characterized, and moreover, various mutant aptamer forms were generated to figure out sequence and structure requirements of the aptamer. Then, we found that intrinsic structure formation and sequence composition of the selected RNA aptamer specific to the antibody are required for the aptamer activity to inhibit the myasthenic autoantibody-mediated destruction of cell surface AChRs. Noticeably, we identified 47-mer minimized aptamer version, which can efficiently protect cells from the effects of the autoantibodies and could be optimally applicable for MG therapy.

Oligonucleotides (ODN) are key molecules for the aim of preventing translation of a gene product or monitoring gene expression in tissues. However, multiple methodological and biological hurdles need to be solved before in vivo application in humans will be possible. For positron emission tomography (PET) investigations, a 20-mer DNA-locked nucleic acid (LNA) mixmer ODN specific for rat chromogranin-A mRNA was labeled with (68)Ga and its uptake was examined in vivo in rats with and without blocking of scavenger receptors by polyribonucleotides. In addition, uptake studies of (68)Ga-LNA were performed with respect to time and concentration in human and rat cell lines. The human cell lines did not express the target mRNA. Both polyinosinic acid (poly-I) and polyadenylic acid (poly-A) reduced the uptake in rat tissues and in human cell lines. Poly-I was found to be more effective in the liver whereas poly-A was more effective in the kidney. In addition, the blockade by poly-I was statistically significant in the pancreas, adrenal gland, bone marrow, intestine, testis, urinary bladder, muscle, parotid gland, and heart, whereas poly-A also caused significant reduction in pancreas, adrenal gland, and bone marrow but not as much as in kidney. Cell culture study showed a 2-phase dose-dependent uptake characteristic with a saturable and a passive diffusion-like phase; however, these 2 phases were not so well expressed in the rat cell line. The results suggest that scavenger receptors or other saturable processes unrelated to hybridization may be involved in the tissue uptake of (68)Ga-LNA and in the clearance of antisense ODN through the liver, kidney, spleen, and bone marrow. The fact that these processes may be sequence-dependent suggests that proof of in vivo hybridization through imaging may not be obtained by only comparing sense and antisense sequences and proving dose-dependency.

Deoxyribozymes (DXZs) are small oligodeoxynucleotides capable of mediating phosphodiester bond cleavage of a target RNA in a sequence-specific manner. These molecules are a new generation of artificial catalytic nucleic acids currently used to silence many disease-related genes. The present study describes a DXZ (Dz1023-434) directed against the polycistronic mRNA from the E6 and E7 genes of human papillomavirus type 16 (HPV-16), the main etiological agent of cervical cancer. Dz1023-434 showed efficient cleavage against a bona fide antisense window at nt 410-445 within HPV-16 E6/E7 mRNA even in low [Mg(2+)] conditions. Using a genetic analysis as guidance, we introduced diverse chemical modifications within Dz1023-434 catalytic core to produce a stable locked nucleic acid (LNA)-modified DXZ (Dz434-LNA) with significant cleavage activity of full E6/E7 transcripts. Cell culture testing of Dz434-LNA produced a sharp decrement of E6/E7 mRNA levels in HPV-16-positive cells resulting in decreased proliferation and considerable cell death in a specific and dose-dependent manner. No significant effects were observed with inactive or scrambled control DXZs nor from using HPV-negative cells, suggesting catalysis-dependent effect and high specificity. The biological effects of Dz434-LNA suggest a potential use for the treatment of cervical cancer.

Using whole living cells as a target for SELEX (systematic evolution of ligands by exponential enrichment) experiments represents a promising method to generate cell receptor-specific aptamers. These aptamers have a huge potential in diagnostics, therapeutics, imaging, regenerative medicine, and target validation. During the SELEX for selecting DNA aptamers, one important step is the separation of 2 DNA strands to yield one of the 2 strands as single-stranded DNA aptamer. This is being done routinely by biotin labeling of the complementary DNA strand to the desired aptamer and then separating the DNA strand by using streptavidin-coated magnetic beads. After immobilization of the double-stranded DNA on these magnetic beads and alkaline denaturation, the non-biotinylated strand is being eluted and the biotinylated strand is retarded. Using Western blot analysis, we demonstrated the detachment of covalent-bonded streptavidin from the bead surface after alkaline treatment. The eluates were also contaminated with undesired biotinylated strands. Furthermore, a streptavidin-induced aggregation of target cells was demonstrated by flow cytometry and microscopic methods. Cell-specific enrichment of aptamers was not possible due to clustering and patching effects triggered by streptavidin. Therefore, the use of streptavidin-coated magnetic beads for DNA strand separation should be examined thoroughly, especially for cell-SELEX applications.

The analysis of microorganism communities in uncultured environmental samples requires laborious and cumbersome techniques such as denaturing gradient gel electrophoresis of amplicons generated with 16S rRNA generic primers with subsequent fragment sequencing. We have developed a simple method for genus identification of methanogen archaea and sulfate-reducing bacteria based on a real-time PCR hybridization probe melting curve analysis. The method takes advantage of a recent explosion of microorganism sequencing data conveniently packaged in the Ribosomal Database Project. Specificity of detection is based on a genus-specific real-time PCR fluorescent 5'-MGB-probe melt. As the probes are designed to have destabilizing mismatches with undesired genera, only samples with a proper melting temperature are called positive.

Targeted gene repair directed by single-stranded oligodeoxynucleotides (ssODNs) offers a promising tool for biotechnology and gene therapy. However, the methodology is currently limited by its low frequency of repair events, variability, and low viability of "corrected" cells. In this study, we showed that during ssODN-mediated gene repair reaction, a significant population of corrected cells failed to divide, and were much more prone to undergo apoptosis, as marked by processing of caspases and PARP-1. In addition, we found that apoptotic cell death triggered by ssODN-mediated gene repair was largely independent of the ATM/ATR kinase. Furthermore, we examined the potential involvement of the mismatch repair (MMR) proteins in this "correction reaction-induced" cell death. Result showed that while defective MMR greatly enhanced the efficiency of gene correction, compromising the MMR system did not yield any viable corrected clone, indicating that the MMR machinery, although plays a critical role in determining ssODN-directed repair, was not involved in the observed cellular genotoxic responses.

The serine protease inhibitor plasminogen activator inhibitor-1 (PAI-1) is increased in several cancers, including breast, where it is associated with a poor outcome. Metastatic breast cancer has a dismal prognosis, as evidenced by treatment goals that are no longer curative but are largely palliative in nature. PAI-1 competes with integrins and the urokinase plasminogen activator receptor on the surface of breast cancer cells for binding to vitronectin. This results in the detachment of tumor cells from the extracellular matrix, which is critical to the metastatic process. For this reason, we sought to isolate RNA aptamers that disrupt the interaction between PAI-1 and vitronectin. Through utilization of combinatorial chemistry techniques, aptamers have been selected that bind to PAI-1 with high affinity and specificity. We identified two aptamers, WT-15 and SM-20, that disrupt the interactions between PAI-1 and heparin, as well as PAI-1 and vitronectin, without affecting the antiprotease activity of PAI-1. Furthermore, SM-20 prevented the detachment of breast cancer cells (MDA-MB-231) from vitronectin in the presence of PAI-1, resulting in an increase in cellular adhesion. Therefore, the PAI-1 aptamer SM-20 demonstrates therapeutic potential as an antimetastatic agent and could possibly be used as an adjuvant to traditional chemotherapy for breast cancer.

Canonical small interfering RNA (siRNA) duplexes are potent activators of the mammalian innate immune system. The induction of innate immunity by siRNA is dependent on siRNA structure and sequence, method of delivery, and cell type. Synthetic siRNA in delivery vehicles that facilitate cellular uptake can induce high levels of inflammatory cytokines and interferons after systemic administration in mammals and in primary human blood cell cultures. This activation is predominantly mediated by immune cells, normally via a Toll-like receptor (TLR) pathway. The siRNA sequence dependency of these pathways varies with the type and location of the TLR involved. Alternatively nonimmune cell activation may also occur, typically resulting from siRNA interaction with cytoplasmic RNA sensors such as RIG1. As immune activation by siRNA-based drugs represents an undesirable side effect due to the considerable toxicities associated with excessive cytokine release in humans, understanding and abrogating this activity will be a critical component in the development of safe and effective therapeutics. This review describes the intracellular mechanisms of innate immune activation by siRNA, the design of appropriate sequences and chemical modification approaches, and suitable experimental methods for studying their effects, with a view toward reducing siRNA-mediated off-target effects.

Nuclear magnetic resonance (NMR) studies have shown that RNA/DNA oligomers with GGA repeat sequences contain unique G-quadruplex structures in the presence of K(+) or Na(+) ions. In this study, we used microchip electrophoresis to study the structure of an RNA aptamer against bovine prion protein that possessed four GGA-triplet repeats (wt2). We analyzed the structural changes and characterized dimer formation of the aptamer. Mutational, circular dichroism, and one-dimensional NMR studies of wt2 revealed that K(+) ions induce wt2 to assume a thermostable dimer in an intramolecular G-quadruplex with parallel orientation.