Oligonucleotides最新文献

The exploration of messenger RNA (mRNA) as a potential therapeutic regulator of gene expression has been significantly reduced by the inability of polyplexes to escape the endocytic pathway, combined with the lack of specific targeting. In the present study, we have developed a site-specific delivery strategy for mRNA molecules through the use of photochemical internalization (PCI) technology. When using EGFP mRNA as a model system, a 10- to 40-fold increase in EGFP-positive cells was obtainable in PCI-treated samples, compared to untreated PCI samples in a human osteosarcoma cell line. The amount of EGFP-positive cells in both PCI and non PCI-treated samples were highly dependent on the nitrogen/phosphate (N/P) ratio. Potent delivery of mRNA molecules through the endocytic pathway by the use of polyplexes and PCI was achievable without any loss of cell viability. The main benefit of the strategy proposed is the possibility for protein production from the delivered mRNA in a way that is controllable in a time- and site-specific manner.

Influenza viruses A and B cause widespread infections of the human respiratory tract; however, existing vaccines and drug therapy are of limited value for their treatment. Here, we show that bispecific short-hairpin small-interfering RNA constructs containing an 8-nucleotide intervening spacer, targeted against influenza virus A or influenza virus B, can inhibit the production of both types of virus in infected cell lines. This multiple vector showed remarkable ability to cope with both influenza viruses A and B. Furthermore, the Autographa californica multiple nuclear polyhedrosis virus can infect a range of mammalian cells, facilitating its use as a baculovirus vector for gene delivery into cells. In this study, baculovirus-mediated bispecific short-hairpin RNA expression markedly inhibited both influenza viruses A and B production.

Silencing genes essential for replication and division using siRNA has potential as a therapeutic strategy for cancer treatment. In order to identify the most potent siRNA, target sequence and siRNA design must be considered together, as tolerance for structural changes can be sequence-dependent. Here we have used growth inhibition assays to investigate the effects of silencing of RRM1, RRM2, and PLK1 with standard siRNAs, Stealth() duplexes, and Dicer substrate siRNAs. The growth inhibitory effect of RRM1, RRM2, or PLK1 knockdown in A549 cells varied with mRNA target site and the format of the siRNA, with longer modified siRNAs generally more effective than standard siRNAs specific for the same target site. Standard siRNAs of varying activity became more potent inhibitors of growth when converted to Stealth() duplexes, and the increase in activity was due to a combination of chemical modification and length. In each case, the effect on activity of changing the siRNA format depended on the siRNA sequence. Taken together these results suggest that, in vitro, longer siRNAs with chemical modifications are in general more active than standard siRNAs targeting the same site, and that structure, chemical modification, and target site must be considered together to identify the most active siRNAs.

Although RNA interference (RNAi) has emerged as an important tool for studying the effects of gene knockdown, it is still difficult to predict the success of RNAi effectors in human systems. By examining the basic thermodynamic equations for RNA interactions in RNAi, we demonstrate how the free energies of RNA folding and phosphoester bond hydrolysis can drive RNAi without ATP. Our calculations of RNAi efficiency are close to actual values obtained from in vitro experimental data from 2 previous studies, for both silencing complex formation (2.50 vs. 2.40 for relative efficiency of RISC formation) and mRNA cleavage (0.50 vs. 0.56 for proportion cleaved). Our calculations are also in agreement with previous observations that duplex unwinding and target site folding are major energy barriers to RNAi.

The overt loss or uncontrolled gain of gene expression is found at some level in virtually every malady afflicting humans. From cancer to HIV-1, the uncontrolled expression or loss of gene expression is prevalent in human diseases. Approaches toward the specific control of gene expression at the transcriptional level could have the potential to revert or reduce disease pathologies. Over the last several years, researchers have developed methodologies that utilize small antisense non-coding RNAs to specifically silence transcription. Only recently has the endogenous molecular pathway usurped by the introduction of these small RNAs to regulate transcription in human cells been defined. Observations suggest that long antisense non-coding RNAs function as the endogenous epigenetic regulators of transcription in human cells, thus explaining why small antisense RNAs were observed early on to silence transcription via directed epigenetic changes at the target loci. The mechanism of action whereby small regulatory RNAs can either turn gene transcription on or off will be discussed as evidence that one day it may be possible to develop therapeutics to regulate gene transcription and ameliorate particular disease conditions.

Several minor groove binding agents (MGBD) were synthesized to study their binding behaviors and sequence specificity with DNA. In order to further understand the binding interactions of the MGBD to DNA, we have synthesized some novel benzimidazoles, which have electron donating (OCH(3), OCH(2)CH(3), OH, O(CH(2))(3)NH(2)) and electron withdrawing cyano groups on the phenyl ring. The interaction of these new benzimidazoles along with parent compounds Hoechst 33342 have been studied with CT DNA, two A-T rich [d(GA(5)T(5)C) and d(CGCA(3)T(3)G)] and one G-C rich [d(GCATGGCCATGC)] oligonucleotide sequences using electrospray ionization mass spectrometry (ESI-MS), absorption, fluorescence, and circular dichroism (CD) spectroscopy. Bisubstituted analogs, which have electron-donating groups, were found to form more stable ligand-DNA complex than Hoechst 33342, while the benzimidazole with electron withdrawing cyano group resulted comparatively in less stable ligand DNA complex. The ESI-MS also gave reliable information about the A-T sequence selectivity as we did not observe any signal with G-C sequence in mass with parent as well as novel ligands. Similar studies with ESI-MS suggest that Hoechst 33342, ETBBZ, and MMBBZ form complexes of 2:1 stoichiometry with d(GA(5)T(5)C) duplex while rest of the ligands form complexes of 1:1 stoichiometry with d(GA(5)T(5)C). Thus, this present study provides the rationalization for the difference in binding behaviors of minor groove binding benzimidazole analogs having different substitution on the phenyl ring.

The aim of this study was to identify and to characterize a highly active anti-HIV ribozyme. Therefore, the genome of HIV-1 IIIb was screened for not yet addressed GUC triplets within highly conserved sequences. Here we report the in vitro characteristics and the antiviral activity of the fittest identified anti-HIV hammerhead ribozyme, targeting the 13th GUC triplet within the HIV-1 pol gene (HHPol13). Multiple turnover kinetics were determined in vitro and revealed very promising kinetic data: V(max) = 39 nM/minute, K(m) = 576 nM, k(cat) = 3.9/minute, and K(cat)/K(m) = 6.8/minute/microM. To analyze its antiviral activity the hammerhead ribozyme was expressed retrovirally in Hut78 cells followed by HIV-1 infection. The newly identified ribozyme conferred a long-term inhibition of HIV-1 replication until the end of the observation period at day 56. We were able to demonstrate that the antiviral activity was mainly due to a ribozyme effect combined with a limited antisense activity. Additionally, the effect of the identified ribozyme was compared with a retrovirally expressed siRNA directed against the same target in the HIV-1 pol gene. This siRNA (siPol13) showed no inhibition of HIV replication. In summary, the hammerhead ribozyme HHPol13 was demonstrated to confer superior cleavage and antiviral activity against HIV-1. These results suggest that even in the RNAi era ribozymes still have the potential as highly active antiviral agents.

Structured single-stranded nucleic acids, or aptamers, bind target molecules with high affinity and specificity, which translates into unique therapeutic possibilities. Currently, aptamers can be identified to most proteins, including blood-clotting factors, cell-surface receptors, and transcription factors. Chemical modifications to the oligonucleotides enhance their pharmacokinetics and pharmacodynamics, thus extending their therapeutic potential. Several aptamers have entered the clinical pipeline for applications and diseases such as macular degeneration, coronary artery bypass graft surgery, and various types of cancer. Furthermore, the functional repertoire of aptamers has expanded with the descriptions of multivalent agonistic aptamers and aptamers-siRNA chimeras. This review highlights those aptamers and aptamer-based approaches with particular likelihood of achieving therapeutic application.

Prostate cancer is the most frequently diagnosed malignancy in men. As cancer progresses from an androgen-sensitive stage to hormone-refractory stage, it turns resistant to androgen ablation therapy. At this stage, effective newer therapies that induce apoptosis are needed for treatment of prostate cancer. DNA oligonucleotides homologous to the telomere 3' overhang (T-oligo) induce apoptosis in several human cancer cells. In the present study, we studied the effect of T-oligo on prostate cancer cells. Our studies showed that androgen-independent DU-145 cells are sensitive to T-oligo in terms of inhibition of proliferation. Moreover, T-oligo induced DU-145 cells to undergo apoptosis. Therefore, our results are encouraging for further investigation in the potential application of T-oligo as a novel therapeutic approach for prostate cancer, especially the androgen-independent.