Showing papers in "Oligonucleotides in 2003"
TL;DR: The classic problems encountered during thermal denaturation experiments are presented and it is demonstrated that a number of useful pieces of information can be extracted from these experimental curves.
Abstract: Tm is defined as Temperature of melting or, more accurately, as temperature of midtransition. This term is often used for nucleic acids (DNA and RNA, oligonucleotides and polynucleotides). A thermal denaturation experiment determines the stability of the secondary structure of a DNA or RNA and aids in the choice of the sequences for antisense oligomers or PCR primers. Beyond a simple numerical value (the Tm), a thermal denaturation experiment, in which the folded fraction of a structure is plotted vs. temperature, yields important thermodynamic information. We present the classic problems encountered during these experiments and try to demonstrate that a number of useful pieces of information can be extracted from these experimental curves.
661 citations
TL;DR: The practical utility of short hairpin siRNA bispecific constructs synthesized as a single transcript is demonstrated and it is now possible to introduce promising multivalent siRNA constructs into retroviral and lentiviral vectors for in vivo gene therapeutic applications.
Abstract: Exploiting the phenomenon of RNA interference (RNAi), recent studies established the utility of monospecific small interfering RNAs (siRNAs) in suppressing HIV-1 infection. However, because of the high mutation rate of the HIV genome, there are considerable challenges in the design of fully efficacious gene therapeutic constructs. Therefore, approaches that simultaneously target different stages of the viral life cycle are desirable. In our current studies, we designed bispecific siRNA constructs against HIV-1 cell surface receptors to inhibit viral entry. Dual specific short hairpin siRNA constructs, containing an 8-nucleotide intervening spacer, targeted against either CXCR4 and CD4 or CCR5 and CXCR4 were synthesized by in vitro transcription. Cleavage of the bispecific constructs yielding monospecific siRNAs was shown to occur in cell extracts. Magi-CXCR4 and CCR5 cells transfected with bispecific siRNAs showed significant downregulation of their respective coreceptors, as determined by FACS analysis. This suggested that combinatorial constructs comprising multiple effector motifs were processed in transfected cells into their respective functional siRNAs. Transfected cells were challenged with either X4 (NL4-3) or R5-tropic (BaL-1) strains of HIV-1. Downregulation of the cell surface receptors coincided with resistance to in vitro viral challenge in both Magi cell lines and peripheral blood mononuclear cells (PBMCs). These results demonstrated the practical utility of short hairpin siRNA bispecific constructs synthesized as a single transcript. Because the short hairpin design will permit tandem assembly of multiple effector motifs, it is now possible to introduce promising multivalent siRNA constructs into retroviral and lentiviral vectors for in vivo gene therapeutic applications.
172 citations
TL;DR: Some strategies for solving some of the problems of small interfering RNA interference are presented, including the development of genetically stable and highly active siRNA expression vectors, a procedure for selection of favorable target sites, and an efficient and inexpensive procedure for constructing an si RNA expression library.
Abstract: RNA interference (RNAi) is a phenomenon whereby expression of an individual gene is specifically silenced by the introduction of a double-stranded RNA (dsRNA) whose sequence is homologous to that of the gene in question. The generation of a small interfering RNA (siRNA) expression library directed against the entire human genome is a project that requires solutions to many difficult technical problems. We present here some strategies for solving some of these problems, including the development of genetically stable and highly active siRNA expression vectors, a procedure for selection of favorable target sites, and an efficient and inexpensive procedure for constructing an siRNA expression library.
111 citations
TL;DR: The start codon region in E. coli mRNA is the most reliable target site for antisense PNAs, and the sensitivity of this region should hold true for most bacterial genes as well as for other RNase H-independent antisense agents that rely on a steric blocking mechanism.
Abstract: Antisense peptide nucleic acids (PNA) can inhibit bacterial gene expression with gene and sequence specificity. Using attached carrier peptides that aid cell permeation, the antisense effects when targeting essential genes are sufficient to prevent growth and even kill bacteria. However, many design uncertainties remain, including the difficult question of target sequence selection. In this study, we synthesized 90 antisense peptide-PNAs to target sequences in a head to tail manner across the entire length of the mRNA encoding beta-lactamase. The results from this scan pointed to the start codon region as most sensitive to inhibition. To confirm and refine the result, a higher-resolution scan was conducted over the start codon region of the beta-lactamase gene and the essential Escherichia coli acpP gene. For both genes, the start codon region, including the Shine-Dalgarno motif, was sensitive, whereas antisense agents targeted outside of this region were largely ineffective. These results are in accord with natural antisense mechanisms, which typically hinder the start codon region, and the sensitivity of this region should hold true for most bacterial genes as well as for other RNase H-independent antisense agents that rely on a steric blocking mechanism. Therefore, although other design parameters are also important, the start codon region in E. coli mRNA is the most reliable target site for antisense PNAs.
108 citations
TL;DR: The dendrimers were found to be successful mediators of transfection of the HeLa cells with a DNA plasmid containing the functional gene of enhanced green fluorescent protein (EGFP), but they failed to do so in HUVEC cell culture.
Abstract: A series of water-soluble polycationic dendrimers with a phosphoramidothioate backbone (P-dendrimers) was studied in human cell culture. Preliminary studies have shown that P-dendrimers of series 1 and 2, possessing N,N-diethyl-ethylenediamine hydrochloride functions at the surface, show rather moderate cytotoxicity toward HeLa, HEK 293, and HUVEC cells in a standard MTT assay in serum-containing medium, generally lower than lipofectin. The experiments of cellular uptake have shown the necessity for the presence of serum for transfection with P-dendrimers of series 1 and 2. These compounds efficiently delivered fluorescein-labeled oligodeoxyribonucleotide into HeLa cells in serum-containing medium, but they failed to do so in HUVEC cell culture. The dendrimers were found to be successful mediators of transfection of the HeLa cells with a DNA plasmid containing the functional gene of enhanced green fluorescent protein (EGFP).
106 citations
TL;DR: Tandem LC-MS analysis confirmed the identity of the oligonucleotide metabolites, failure products, the presence of protection groups not removed after synthesis, and the extent of depurination or phosphorothioate backbone oxidation.
Abstract: A method for the analysis and characterization of therapeutic and diagnostic oligonucleotides has been developed using a combination of liquid chromatography and mass spectrometry (LC-MS). The optimized ion-pairing buffers permit a highly efficient separation of native and chemically modified antisense oligonucleotides (AS-ODNs) from their metabolites or failure synthetic products. The mobile phases were MS compatible, allowing for direct and sensitive analysis of components eluting from the column. The method was applied for the quantitation and characterization of AS-ODNs, including phosphorothioates and 2 9-O-methyl-modified phosphorothioates. Tandem LC-MS analysis confirmed the identity of the oligonucleotide metabolites, failure products, the presence of protection groups not removed after synthesis, and the extent of depurination or phosphorothioate backbone oxidation.
88 citations
TL;DR: A key role for Bcl-2 in conferring chemoresistance to melanoma is underline and Bcl2 siRNA strategies are highlighted as novel and highly effective tools, with the potential for future targeted therapy of malignant melanoma.
Abstract: Malignant melanoma is a prime example of a treatment-resistant tumor with poor prognosis. Even with innovative treatment regimens, response rates remain low, and the duration of responses is short....
84 citations
TL;DR: It is demonstrated that the sense of Pchirality of PS-oligos plays a major role in determining the biologic activities of CpG motifs, but Sp-chiralities of the PS-OLigo appears to improve stability and may provide more durable effects in prolonged tissue culture systems.
Abstract: Many of the biologic activities of phosphorothioate oligodeoxynucleotides (PS-oligos) are affected by the sense of chirality of the phosphorus atoms of the internucleotide linkages. Some of the activities are increased by the Rp stereoisomer, and others are increased by the Sp stereoisomer. In previous studies, we showed that PS-oligos containing unmethylated CpG dinucleotides in particular sequence contexts can stimulate B cells and other immune cells. These CpG PS-oligos trigger mitogenactivated protein kinase (MAPK) signaling pathways, causing the induction of B cell proliferation and cytokine and immunoglobulin secretion. We investigated whether the immune stimulation by CpG PS-oligos depends on the sense of their P-chirality. CpG PS-oligos synthesized with internucleotide phosphorothioates of Rp configuration at P-atom showed much stronger MAPK activation and induction of I kappa B degradation after 40 minutes of stimulation compared with PS-oligos synthesized with Sp linkages. In order to determine if the enhanced stimulatory effects of the Rp stereoisomer may result from differential cellular uptake, we examined the rates at which fluorescently labeled Rp or Sp CpG PS-oligos were taken up by B cells, but these were found to be identical to each other and to stereorandom PS-oligos. The stronger stimulatory effect of the R stereoisomer did not last for 48 hours, and (3)H-thymidine incorporation assays at this point showed that only the S stereoisomer was active--to approximately the same level as induced by PS-oligos with stereorandom phosphorothioate linkages. This loss of activity of the R stereoisomer most likely resulted from rapid degradation of the oligonucleotides rather than from reduced interaction with the CpG receptor because PS-oligos in which only the CpG dinucleotide was stereodefined were most stimulatory when the CpG was Rp but not when the CpG was Sp. These studies demonstrate that the sense of Pchirality of PS-oligos plays a major role in determining the biologic activities of CpG motifs. Rp-chirality at the CpG is preferred for best stimulation at early time points, but Sp-chirality of the PS-oligo appears to improve stability and may provide more durable effects in prolonged tissue culture systems.
61 citations
TL;DR: This work engineered stably transfected human cells that express large dsRNAs mediating specific posttranscriptional silencing of genes, and used this technique to specifically silence genes coding for glucosylceramide synthase, the sphingolipid activator protein precursor (SAP), and glucocerebrosidase, all implicated in glycosphingolIPid metabolism.
Abstract: Recently, double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) has rapidly developed to a powerful instrument for specific silencing of gene expression in several organisms, including Caenorhabditis elegans, Drosophila melanogaster, and plants. The finding that synthetic small interfering RNAs (siRNAs) of 21 nt as well as stable, endogenously expressed, large dsRNA are suited to specifically induce gene silencing in mammalian cells offered the possibility of expanding this technique to mammalian systems. In this work, we engineered stably transfected human cells that express large dsRNAs mediating specific posttranscriptional silencing of genes. We used this technique to specifically silence genes coding for glucosylceramide synthase (GCS), the sphingolipid activator protein precursor (SAP), and glucocerebrosidase (GBA), all implicated in glycosphingolipid metabolism. From a 1600-bp inverted repeat DNA template, a dsRNA of 800 bp is expressed and predicted to mediate the specific suppression of the corresponding gene by RNAi. Remarkably, we were able to use this method to achieve complete inhibition of those genes we targeted in different cultured human cell lists. These findings testify to the generality of RNAi application in suppressing gene expression in mammalian cells.
57 citations
TL;DR: A striking aspect of these results is that such marked differences in CpG-ODN induced innate responses existed both between and within two closely related species.
Abstract: Cytosine-phosphate-guanosine (CpG)-DNA can induce an impressive array of innate immune responses that may directly or indirectly contribute to the clearance of infectious agents. Assays, such as lymphocyte proliferative responses, have been used to demonstrate that the immunostimulatory activity of CpG-DNA is conserved among a broad range of vertebrate species, but no studies have been completed to determine if qualitative differences exist among species for CpG-oligodeoxynucleotide (ODN)-induced innate immune responses. In this study, we assessed the capacity of a Class A (ODN 2216) and a Class B (ODN 2007) CpG-ODN to induce innate immune responses in two closely related species, ovine (n = 28) and bovine (n = 29). The secretion of interferon (IFN)-α and IFN-γ and non-major histocompatability complex (MHC)-restricted cytotoxic activity were assayed with CpG-ODN-stimulated peripheral blood mononuclear cells (PBMC). These investigations revealed significant interspecies and intraspecies variation in the re...
54 citations
TL;DR: A strategy to transduce phagocytic cells, reduce cell membrane CCR5, and protect from infection with R5-tropic HIV using rSV40-delivered, VA promoter-driven siRNAs is described.
Abstract: Transducing macrophages and other phagocytic cells has been problematic because these cells are largely nondividing and can phagocytose and degrade viral gene delivery vectors. Because of their carriage of the CCR5 chemokine receptor that functions as a coreceptor for most clinical strains of HIV, these cells are also key targets in early HIV infection and dissemination. We describe here a strategy to transduce these phagocytes, reduce cell membrane CCR5, and protect from infection with R5-tropic HIV. Recombinant Tag-deleted SV40 vectors were used to transduce unselected CCR5-bearing cell lines and primary cells with >98% efficiency. rSV40s were designed to express two different anti-CCR5 small interfering RNAs (siRNAs), driven by the adenoviral VA1 polymerase III (pol III) promoter, which localizes the transcripts in the cytoplasm. Transduction with both siRNAs substantially reduced CCR5 mRNA, which in turn decreased detectable cell membrane CCR5. Both CCR5 + cell lines and primary cells were used: SupT1...
TL;DR: The fact that a DNA aptamer could interfere with the binding of natural templates of the enzyme could help in performing structure-function analysis of the NS5B and might constitute a basis for further structure-based drug design of this crucial enzyme of HCV replication.
Abstract: The RNA-dependent RNA polymerase (NS5B) of the hepatitis C virus (HCV) plays a key role in the life cycle of the virus. In order to find inhibitors of the HCV polymerase, we screened a library of 8...
TL;DR: OMe/LNA mixmers are powerful reagents for use as steric block inhibitors of gene expression regulated by protein-RNA interactions within HeLa cell nuclei.
Abstract: The HIV-1 trans-activation responsive element (TAR) RNA stem-loop interacts with the HIV trans-activator protein Tat and other cellular factors to stimulate transcriptional elongation from the viral long terminal repeat (LTR). Inhibitors of these interactions block full-length transcription and, hence, would potentially inhibit HIV replication. We have studied structure-activity relationships in inhibition of trans-activation by steric block 2′-O-methyl (OMe) oligonucleotides chimeras (mixmers) containing locked nucleic acid (LNA) units. Inhibition was measured both in Tat-dependent in vitro transcription from an HIV-1 DNA template directed by HeLa cell nuclear extract and in a robust HeLa cell reporter assay that involves use of stably integrated plasmids to express firefly luciferase Tat dependently and Renilla luciferase Tat-independently. OMe oligonucleotides with optimally 40%-50% LNA units and a minimum of 12 residues in length were active in the cellular assay when delivered with cationic gemini su...
TL;DR: The results demonstrate the feasibility of using a lentiviral vector to stably transduce therapeutic shRNAs into leukemia cells for the potential ex vivo purging of Ph+ cells in an autologous hematopoietic cell transplant setting and suggests that this system could be generally useful for the expression of other sh RNAs.
Abstract: Chronic myeloid leukemia (CML) is characterized by a reciprocal chromosomal translocation between chromosomes 9 and 22 t(9;22)(q34;q11) that causes fusion of the bcr and abl genes. Transcription and splicing of the fusion gene generate two major splice variants of the bcr/abl transcript that encode an oncoprotein with tyrosine kinase activity. We have taken advantage of lentiviral vectormediated delivery of anti-bcr/abl short hairpin RNAs (shRNA) to downregulate the bcr/abl transcript in Philadelphia chromosome-positive (Ph+) K562 leukemia cells. This downregulation caused complete inhibition of proliferation of these cells and was accompanied by >90% inhibition of the bcr/abl transcript and p210 protein. These results demonstrate the feasibility of using a lentiviral vector to stably transduce therapeutic shRNAs into leukemia cells for the potential ex vivo purging of Ph+ cells in an autologous hematopoietic cell transplant setting. Furthermore, the robust expression of the shRNAs from our lentiviral vector suggests that this system could be generally useful for the expression of other shRNAs.
TL;DR: Lentiviral gene transfer of shRNA expression cassettes may be used to induce long-term RNAi in human hematopoietic stem and progenitor cells for functional genetics and potential therapeutic intervention.
Abstract: RNA interference (RNAi) describes a highly conserved mechanism of sequence-specific posttranscriptional gene silencing triggered by double-stranded RNA (dsRNA). Whereas RNAi is applied to study gene function in different organisms and in variant cell types, little is known about RNAi in human hematopoietic stem and progenitor cells and their myeloid progeny. To address this issue, short hairpin RNAs (shRNA) were designed to target the common beta-chain of the human receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 (betaGMR). These receptors regulate proliferation, survival, differentiation, and functional activity of hematopoietic cells. In addition to markedly inhibiting mRNA and protein expression, anti-beta-GMR shRNAs were also found to inhibit receptor function in a cell culture model. Furthermore, lentiviral gene transfer of shRNA expression cassettes into primary normal CD34+ cells selectively inhibited colony formation of transduced progenitors when stimulated with GM-CSF/IL-3 but not when stimulated with cytokines that do not signal via beta-GMR. Finally, anti-beta-GMR shRNAs had no detectable effect on engraftment or lineage composition of lentivirally transduced human CD34+ cells transplanted into NOD/SCID mice. However, the growth defect of transduced colony-forming cells under stimulation with GM-CSF/IL-3 remains unchanged in bone marrow cells harvested from individual NOD/SCID mice 6 weeks after transplantation. These data indicate that lentiviral gene transfer of shRNA expression cassettes may be used to induce long-term RNAi in human hematopoietic stem and progenitor cells for functional genetics and potential therapeutic intervention.
TL;DR: RNA interference (RNAi) offers a suitable technique to dissect signaling pathways employed by NPM-ALK and may potentially be used to develop siRNA-based gene therapeutic approaches against N PM-ALK-positive lymphomas.
Abstract: The NPM-ALK fusion protein is found in up to 75% of pediatric anaplastic large cell lymphomas (ALCL). The ALK kinase becomes constitutively activated and triggers malignant transformation. Molecular targeting of the tumor-specific NPM-ALK fusion by gene-silencing methods seems to be a promising approach both for the treatment of ALCL and to decipher signaling pathways used by NPM-ALK. We designed and evaluated three chemically synthesized small interfering RNAs (siRNAs) for downregulation of the NPM-ALK fusion mRNA. Compared to HeLa cells transfected with the NPM-ALK expression plasmid only and to an siRNA containing two point mutations, the most potent anti-NPM-ALK siRNA reduced NPM-ALK protein expression in HeLa cells to almost undetectable levels, and the number of cells stained positively for NPM-ALK decreased by 80%. With respect to signaling, expressing of NPM-ALK increased the activity of AKT and ERK in HeLa cells, and this effect could be blocked by the specific siRNA targeting NPM-ALK. Expression of endogenous NPM-ALK mRNA in SR786 ALCL cells decreased by 50%-60% in cells transfected with the NPM-ALK siRNA. However, the amount of NPM-ALK protein was not influenced by a single transfection of the siRNAs against NPM-ALK. Repeated transfections over 8 days led to a significant reduction in NPM-ALK protein but without induction of apoptosis. We believe that the long protein half-life of NPM-ALK, at least 48 hours, limits the application of transiently transfected siRNAs. Nevertheless, RNA interference (RNAi) offers a suitable technique to dissect signaling pathways employed by NPM-ALK and may potentially be used to develop siRNA-based gene therapeutic approaches against NPM-ALK-positive lymphomas.
TL;DR: The results obtained with mixed CeNA sequences warrant antisense studies with CeNA and excellent mismatch discrimination has been observed as well for the duplex with DNA as for the Duplex with RNA.
Abstract: Cyclohexene nucleic acids (CeNA) with a D-like configuration form very stable self-complementary duplexes and stable duplexes with RNA. An increased duplex stability with ΔTm/mod of +1.2°C is observed. The duplex with DNA is less stable. Excellent mismatch discrimination has been observed as well for the duplex with DNA as for the duplex with RNA. The results obtained with mixed CeNA sequences warrant antisense studies with CeNA. The CeNAs of opposite chirality constitute a self-pairing system on their own, resembling L-RNA sequences.
TL;DR: A highly efficient and specific small interfering (siRNA) for the serine/threonine kinase Pim-1 has been generated that silences the expression of a Pim1-green fluorescent protein (GFP) fusion gene at low nanomolar concentrations (approximately 5 nM).
Abstract: A highly efficient and specific small interfering (siRNA) (PsiR4) for the serine/threonine kinase Pim-1 has been generated that silences the expression of a Pim1-green fluorescent protein (GFP) fusion gene at low nanomolar concentrations ( ,5 nM). Only one of four siRNAs tested against Pim-1 had high potency, whereas the three other siRNAs were completely inefficient up to a concentration of 100 nM. PsiR4 was labeled with Cy3 at the 5 9-end of the sense strand to investigate cellular uptake and localization in living COS-7 and F-11 cells. This modification has only minor effects on the potency of PsiR4 to inhibit Pim1-GFP. Cellular uptake of the Cy3-labeled siRNA by lipofection was observed in more than 90% of the cells and reaches a plateau 4‐ 6 hours after transfection. Cotransfection studies with low PsiR4-Cy3 concentrations demonstrated that most cells that still expressed Pim1-GFP did not show siRNA uptake. Localization studies with PsiR4-Cy3 in the neuronal hybridoma cell line F-11 displayed a dotted, perinuclear accumulation of siRNAs. Moreover, cells with neuritelike structures contain PsiR4 in this cellular compartment.
TL;DR: Fluorescein-labeled siRNAs may lead to false negative results and should not be used to examine electroporation-mediated siRNA uptake, and Cy3 and Cy5- labels cause a significant amount of cell fluorescence, as judged by flow cytometry.
Abstract: Transfection of mammalian cells with preformed small interfering RNAs (siRNAs) permits a transient and often specific reduction of gene expression. It is possible to rapidly examine the uptake of siRNAs by transfection with fluorescently labeled siRNAs. We examined the apparent uptake of such siRNAs by several leukemic cell lines after electroporation. We show that Cy3 and Cy5-labeled siRNAs cause a significant amount of cell fluorescence, as judged by flow cytometry. In contrast, several fluorescein-labeled siRNAs could not be detected. Nevertheless, such fluoresceinated siRNAs efficiently suppressed a leukemic target gene, demonstrating that siRNA uptake must have taken place. Therefore, for cell electroporation, fluorescein-labeled siRNAs may lead to false negative results and should not be used to examine electroporation-mediated siRNA uptake.
TL;DR: It is demonstrated here that very short steric blocking ONs can inhibit the formation of translation preinitiation complexes on the IRES and block IRES-mediated translation in a cell-free translation assay and in a transfected hepatoma cell line.
Abstract: Hepatitis C virus (HCV) infection represents a worldwide problem, and current antiviral regimens are not satisfactory. The need to develop novel, specific, anti-HCV antiviral drugs is clear. Antisense oligonucleotides (AS-ON), ribozymes, and more recently, small interfering RNAs (siRNAs) have been widely used to control gene expression, and several clinical trials are in progress. The potential to use AS-ON as tools to control HCV infection, either by promoting an RNase H mediated cleavage of viral genomic RNA or by interfering with the assembly of a translation initiation complex on the internal ribosome entry site (IRES) is reviewed. Extensive knowledge of IRES structure and conservation among HCV genotypes have rendered the HCV IRES (and, in particular, its IIId loop) particularly attractive for antisense approaches. Encouraging data have been obtained with IRES-targeted RNase H-competent and incompetent ON analogs. We demonstrate here that very short steric blocking ONs can inhibit the formation of translation preinitiation complexes on the IRES and block IRES-mediated translation in a cell-free translation assay and in a transfected hepatoma cell line.
TL;DR: Nonamplifiable detection methods, such as Southern blotting, protein analysis, or functional assays, should be performed, whenever possible, to correctly assess gene conversion frequencies.
Abstract: Recent studies have reported successful correction of the most common F508del mutation in cystic fibrosis (CF) airway epithelial cells by small fragment homologous replacement (SFHR). We wished to apply the SFHR methodology to our CF bronchial epithelial cells, of compound heterozygous genotype (F508del/W1282X), in which nucleic acid transfer was previously optimized by electroporation. Using a PCR-based detection methodology, with one of the primers located outside the SFHR homology region, we obtained SFHR dose-dependent F508del to wild-type CFTR gene conversion frequencies reaching 30%. However, the increased wild-type/F508del CFTR allele ratio was transient, vanishing at 5 days posttransfection. Furthermore, we have been unable to reproduce the SFHR-mediated repair of the F508del mutation in our cellular model when both detection primers were located outside the SFHR homology region. A thorough reexamination of our initial detection strategy revealed that a false positive result was originated from a PCR artifact created by the SFHR fragment itself. Thus, nonamplifiable detection methods, such as Southern blotting, protein analysis, or functional assays, should be performed, whenever possible, to correctly assess gene conversion frequencies.
TL;DR: The chicken embryo has become an efficient in vivo system to study gene function during development and the knowledge of a cDNA fragment of the gene of interest is sufficient to get loss-of-function phenotypes, which is a valuable tool for functional genomics.
Abstract: Genomics has changed the pace by which genes are analyzed. Rather than looking at genes one by one, gene expression today is studied at the genome level. Unfortunately, the data we get from microarray analysis do not give us any clues about the function of these genes. Functional analyses are still refractory to large-scale, high-throughput studies, particularly in vertebrates. With the development of in ovo RNAi as a tool for specific gene silencing, the chicken embryo has become an efficient in vivo system to study gene function during development. A major advantage of in ovo RNAi is the fact that the knowledge of a cDNA fragment of the gene of interest is sufficient to get loss-of-function phenotypes. Thus, this new approach is a valuable tool for functional genomics.
TL;DR: It is found that the all-ribonucleotide siRNA gave the best inhibition of notch expression, in accordance with the hypothesis that primer extension for generation of ssRNA from single-stranded mRNA does not operate in Drosophila.
Abstract: Short interfering RNAs (siRNAs) are the processing product originating from long double-stranded RNAs (dsRNAs) that are cleaved by the RNase III-like ribonuclease Dicer. As siRNAs mediate cleavage of specific single-stranded target RNAs, they are essential intermediates of RNA interference (RNAi). When applied in synthetic form, siRNAs likewise can induce the silencing process in the absence of long dsRNAs. Here, we tested variations of a conventional synthetic siRNA that had been used successfully to silence the Drosophila Notch gene. The variants had two 3 9-terminal deoxynucleotides in their protruding single-stranded ends. In one case, the deoxynulceotides would match to the Notch mRNA, whereas the other variant had nonmatching deoxy-T residues, representing a widely used siRNA design. siRNAs with different combinations of sense and antisense strands were injected into Drosophila embryos at two different concentrations. We found that the all-ribonucleotide siRNA gave the best inhibition of Notch expression. The combination of two modified strands with 3 9-terminal deoxynucleotides was effective, but if combined with a sense or antisense ribostrand, the efficacy dropped. The siRNAs with nonmatching 3 9-terminal TT residues showed a reduced silencing potential, which became evident at low concentration. An siRNA with a nonmatching 39-terminal ribonucleotide in the antisense strand retained most of its silencing potential in accordance with the hypothesis that primer extension for generation of ssRNA from single-stranded mRNA does not operate in Drosophila.
TL;DR: Molecular dynamics studies of the two available structures for the DIS/DIS kissing complex in aqueous solution and in the presence of sodium counterions report a new and simple dimerization process.
Abstract: As in all retroviruses, human immunodeficiency virus (HIV) genomic RNA is packaged into virions as a dimer. The two copies of the genome are noncovalently linked by their 5′-ends in the dimerizatio...
TL;DR: It is found that the siRNAs were somewhat less effective than the S-ODNs in reducing the Raf-1 protein level 20 hours after a 4-hour transfection, adding to others in the literature that show it can be difficult to select si RNAs that are more effective than antisense ODNs in downregulating endogenously expressed proteins.
Abstract: Two sets of 20-mer phosphorothioate-modified oligodeoxynucleotide DNAs (sODN) and 21-mer or 22-mer small interfering RNAs (siRNAs), targeted to the same coding sites in raf-1 mRNA, were compared for their abilities to reduce the amount of endogenously expressed Raf-1 protein in T24 cells. The amount of Raf-1 protein was monitored by careful quantitation of Western blots. We found that the siRNAs were somewhat less effective than the S-ODNs in reducing the Raf-1 protein level 20 hours after a 4-hour transfection. The siRNA duplexes were characterized by circular dichroism (CD) spectra, and melting temperatures (Tm) were obtained for the siRNA duplexes and DNA x RNA hybrids formed by the S-ODNs. The S-ODNs differed in their effectiveness, the S-ODN that formed the more stable hybrid being the more effective in reducing the Raf-1 protein level, but the two siRNAs were equally effective despite a difference in Tm of about 20 degrees C. Finally, the siRNAs and S-ODNs had a comparable nonspecific effect on a nontargeted (Bcl-2) protein. Our data add to others in the literature that show it can be difficult to select siRNAs that are more effective than antisense ODNs in downregulating endogenously expressed proteins.
TL;DR: The remarkable efficacy of the tridecamer PNAs in arresting translation elongation of HIV-1 integrase mRNA is explained by their ability to form stable triplexes at neutral pH with short purine sequences.
Abstract: Recently, we showed that antisense peptide nucleic acids (PNA) containing a short pyrimidine stretch (C4TC3) invade Ha-ras mRNA hairpin structures to form highly stable duplex and triplex complexes...
TL;DR: In this paper, a self-cleaving ribozyme-expressing vector was used to generate small interfering RNAs (siRNAs) in mammalian cells, which can inhibit gene expression in sequence-specific manner without induction of the nonspecific degradation that is activated by long double-stranded RNA (dsRNA) (>30 nt).
Abstract: RNA interference (RNAi) has been developed recently as a powerful tool for silencing of mRNAs in various organisms. In mammalian cells, the introduction of small interfering RNAs (siRNAs) can inhibit gene expression in a sequence-specific manner without induction of the nonspecific degradation that is activated by long double-stranded RNA (dsRNA) (>30 nt). Here, we report a method for generating siRNAs in mammalian cells using a self-cleaving ribozyme-expressing vector. Four ribozymes within transcripts that were expressed under the control of a cytomegalovirus (CMV) or tRNAVal promoter excised, in cis, specific sense and antisense sequences from primary transcripts and generated siRNAs in HeLa cells. The siRNAs generated by the ribozymes were able to decrease the expression of a firefly gene for luciferase. These results suggest that polymerase II (pol II) systems, particularly in view of the availability of many potential tissue-specific promoters, and pol III systems, in which siRNAs are generated by a...
TL;DR: The use of PEG-AS-ODN, affording specific delivery of AS- ODN to target cells, increased cell proliferation, and enhanced ODN uptake, may be of potential importance in stem cell expansion for BM transplantation and gene therapy.
Abstract: To determine whether the efficacy of entry and action of antisense oligonucleotides (AS-ODN) on hematopoietic stem cells in vitro could be improved by the addition of polyethylene glycol (PEG), a m...
TL;DR: AS-ODNs against CD4 molecules inhibited surface and mRNA CD4 expression, under physiologic turnover and, consequently, modulate T CD4+ cell reactivity.
Abstract: In previous studies, we have demonstrated the inhibition of CD4 expression in rat lymphocytes treated with phorbol myristate acetate (PMA) by antisense oligonucleotides (AS-ODNs) directed against the AUG start region of the cd4 gene. The aim of the present study was to inhibit CD4 expression in lymphocytes without promoting CD4 synthesis and to determine the effect of this inhibition on CD4 1 T cell function. Four 21-mer ODNs against the rat cd4 gene (AS-CD4-1 to AS-CD4-4) were used. Surface CD4 expression was measured by immunofluorescence staining and flow cytometry, and mRNA CD4 expression was measured by RT-PCR. T CD4 1 cell function was determined by specific and unspecific proliferative response of rat-primed lymphocytes. After 24 hours of incubation, AS-CD4-2 and AS-CD4-4 reduced lymphocyte surface CD4 expression by 40%. This effect remained for 72 hours and was not observed on other surface molecules, such as CD3, CD5, or CD8. CD4 mRNA expression was reduced up to 40% at 24 hours with AS-CD4-2 and AS-CD4-4. After 48 hours treatment, CD4 mRNA decreased up to 27% and 29% for AS-CD4-2 and AS-CD4-4, respectively. AS-CD4-2 and AS-CD4-4 inhibited T CD4 1 cell proliferative response upon antigen-specific and unspecific stimuli. Therefore, AS-ODNs against CD4 molecules inhibited surface and mRNA CD4 expression, under physiologic turnover and, consequently, modulate T CD4 1 cell reactivity.
Journal Article•
TL;DR: This study identified sites in HIV-2 leader region RNA that are functionally accessible to hybridization with oligonucleotides (ODNs) by reverse transcription with random ODN libraries (RT-ROL) and tested specific ODNs directed against these regions for their efficacy in inhibiting RNA dimerization in vitro.
Abstract: The retroviruses, including the human pathogens HIV-1 and HIV-2, are diploid inasmuch as they encapsidate two copies of their RNA genome. Prior to or during encapsidation, two copies of full-length genomic RNA recognize and stably bind each other in a process called dimerization. RNA structures within the viral genome promote dimerization in both HIV-1 and HIV-2 and are located in the 5'-untranslated leader region. Inhibition of dimerization by mutation of these RNA signals has been demonstrated to drastically reduce viral infectivity and replication kinetics and, thus, represents a potential target for antiretroviral therapy. In this study, we identified sites in HIV-2 leader region RNA that are functionally accessible to hybridization with oligonucleotides (ODNs) by reverse transcription with random ODN libraries (RT-ROL). We then tested specific ODNs directed against these regions for their efficacy in inhibiting RNA dimerization in vitro. We determined that of several hybridization-competent ODNs, only two were very effective in inhibiting RNA dimerization. Both of these ODNs were complementary to viral RNA at the primer binding site (PBS). These results identify regions with high accessibility to ODN binding on HIV-2 RNA and help to map the region(s) essential for dimerization within the viral RNA.