Oligonucleotides最新文献

{"title":"Hyaluronic acid-modified DOTAP/DOPE liposomes for the targeted delivery of anti-telomerase siRNA to CD44-expressing lung cancer cells.","authors":"Sebastian Taetz,&nbsp;Amélie Bochot,&nbsp;Claudio Surace,&nbsp;Silvia Arpicco,&nbsp;Jack-Michel Renoir,&nbsp;Ulrich F Schaefer,&nbsp;Véronique Marsaud,&nbsp;Saadia Kerdine-Roemer,&nbsp;Claus-Michael Lehr,&nbsp;Elias Fattal","doi":"10.1089/oli.2008.0168","DOIUrl":"https://doi.org/10.1089/oli.2008.0168","url":null,"abstract":"<p><p>Cationic hyaluronic acid (HA)-modified DOTAP/DOPE liposomes were designed for the targeted delivery of anti-telomerase siRNA to CD44 receptor-expressing lung cancer cells. DOTAP/DOPE liposomes modified with 1%-20% (w/w) HA-DOPE conjugate were obtained by the ethanol injection method. Their size was below 170 nm and they exhibited zeta potentials higher than +50 mV. Lipoplexes prepared at different +/-ratios with siRNA were in the range of 200 nm and below and their zeta potentials were strongly dependent on the degree of modification and the +/-charge ratio. The presence of HA did not compromise binding, protection of siRNA from degradation, and complex stabilities in serum but rather resulted in an improvement of these properties. Liposome cytotoxicity, investigated by the MTT assay and LDH release after treatment of CD44(+) A549 cells and CD44(-) Calu-3, was demonstrated only at high concentrations. However, the addition of siRNA to HA-modified liposomes prevented cytotoxic effects compared to all other formulations. As shown by flow cytometry, transfection of siRNA into A549 cells was markedly improved with HA-modified liposomes, but not into Calu-3 cells. Using a qPCR-TRAP assay to test telomerase activity, no difference was demonstrated in the efficiency between HA-modified and nonmodified preparations. Moreover, some reduction in telomerase activity was observed with liposomes alone, lipoplexes prepared with nonsense siRNA and lipofectamine, indicative for some direct inhibitory effect of the lipids and siRNA on the expression of this enzyme. HA-modified DOTAP/DOPE liposomes represent a suitable carrier system for siRNA since properties like binding or protection of siRNA are not altered. They display an improved stability in cell culture medium and a reduced cytotoxicity. Furthermore, these novel lipoplexes could successfully be targeted to CD44-expressing A549 cells opening interesting perspectives for the treatment of lung cancer.</p>","PeriodicalId":19523,"journal":{"name":"Oligonucleotides","volume":"19 2","pages":"103-16"},"PeriodicalIF":0.0,"publicationDate":"2009-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/oli.2008.0168","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"28189398","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"siRNA-mediated integrin-linked kinase suppression: nonspecific effects of siRNA/cationic liposome complexes trigger changes in the expression of phosphorylated-AKT and mTOR independently of ILK silencing.","authors":"Maite Verreault,&nbsp;Marcel B Bally","doi":"10.1089/oli.2008.0157","DOIUrl":"https://doi.org/10.1089/oli.2008.0157","url":null,"abstract":"<p><p>Short interfering RNA targeting ILK (ILK siRNA) could be used to treat patients with cancers where constitutive activation of the AKT/PI3K pathway is prominent (e.g., those cancers lack functional PTEN). It is generally believed that siRNA therapeutics will require the use of delivery systems and lipid-based formulations containing cationic lipids (CLs) are a viable option. However, CLs are known to be toxic and exposure to CLs can influence cell survival pathways. This study characterized how CLs combine with ILK siRNA to influence the AKT/PI3K pathway. Using PTEN-negative cell lines (PC3 castration-insensitive prostate cancer cells and U251 glioma cancer cells), the influence of CLs on the downstream consequences of ILK silencing was determined. When comparing nucleofection (an electroporation method that does not require the use of CLs) and CLs as means to deliver ILK siRNA, a 12- to 30-fold increase in siRNA delivery was achieved when using a CL formulation, yet ILK suppression was less efficient. Importantly, time-dependent signaling consequences associated with ILK silencing, including suppression of phosphorylated (serine 473)-AKT and changes in mTOR expression, were observed independently of ILK suppression when the target cells were exposed to cationic lipids following nucleofection-based delivery of ILK siRNA.</p>","PeriodicalId":19523,"journal":{"name":"Oligonucleotides","volume":"19 2","pages":"129-40"},"PeriodicalIF":0.0,"publicationDate":"2009-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/oli.2008.0157","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"28119733","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Antitumor activity of small double-stranded oligodeoxynucleotides targeting telomerase RNA in malignant melanoma cells.","authors":"Faiza Noreen,&nbsp;Jochen Heinrich,&nbsp;Karin Moelling","doi":"10.1089/oli.2008.0170","DOIUrl":"https://doi.org/10.1089/oli.2008.0170","url":null,"abstract":"<p><p>Human telomerase RNA (hTR) is an intrinsic component of telomerase enzyme. Small interfering RNAs (siRNAs) and single-stranded antisense oligonucleotides have been used previously for silencing of the hTR. The objective of this study was to investigate the effect of partially double-stranded oligodeoxynucleotides (ODNs), in vitro and in vivo in comparison to single-stranded antisense ODNs and siRNAs. ODNs were designed on the basis of structural properties of an ODN from previous studies on HIV, to target the hTR in the human cervical carcinoma HeLa cell line and mouse telomerase RNA (mTR) in the murine metastatic melanoma B16-F10 cell line, respectively. Our results indicate that ODNs were able to inhibit the hTR by 68% and the mTR by 81% in the respective cell lines. This correlated with ODN-mediated rapid inhibition of cell proliferation and induction of apoptosis excluding slow effects on telomerase function. The inhibition of the hTR was decreased by knock-down of the cellular RNases H suggesting their contribution. Furthermore, we showed a reduction in numbers of metastases by 70% after intravenous administration of ODN-transfected B16-F10 cells in C57BL/6 mice. Our study demonstrates the potential utility of these hairpin-loop-structured ODNs as a different group of nucleic acids for telomerase-based antiproliferative strategies.</p>","PeriodicalId":19523,"journal":{"name":"Oligonucleotides","volume":"19 2","pages":"169-78"},"PeriodicalIF":0.0,"publicationDate":"2009-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/oli.2008.0170","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"28173712","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optimized basic conditions are essential for successful siRNA transfection into primary endothelial cells.","authors":"Andrea Nolte,&nbsp;Claudia Raabe,&nbsp;Tobias Walker,&nbsp;Perikles Simon,&nbsp;Gerhard Ziemer,&nbsp;Hans Peter Wendel","doi":"10.1089/oli.2009.0182","DOIUrl":"https://doi.org/10.1089/oli.2009.0182","url":null,"abstract":"<p><p>RNA interference (RNAi) is a powerful technique in basic research and has a high potential for therapeutic applications. To realize its clinical applicability, introduction of short double-stranded RNA (dsRNA) has to be carried out under physiological conditions. This study evaluates two cationic liposomal transfection reagents on the efficiency of successful silencing of primary human endothelial cells. Transfection efficiency was investigated under different conditions, for example different media during transfection, duration of transfection, siRNA concentration, and the use of serum and antibiotics. Viability after transfection was examined by CASY and MTT assay. Interferon response was examined by real-time PCR. First we revealed that transfection carried out in the presence of serum and antibiotics caused good knockdown results only by the use of the novel lipid cationic transfection reagent. Both lipid cations had slightly the same transfection efficiency over the range of 10-150 nM siRNA concentration. Examination of interferon response showed increasing OAS1 and STAT1 expression, but not as high as if the transfections were carried out with synthetic polyinosinic-polycytidylic acid double-stranded RNA (poly[IC]). The optimized combination of basic conditions for transfection significantly enhanced the efficiency of the siRNA-mediated knockdown, without causing toxicity or stimulation of the interferon pathway.</p>","PeriodicalId":19523,"journal":{"name":"Oligonucleotides","volume":"19 2","pages":"141-50"},"PeriodicalIF":0.0,"publicationDate":"2009-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/oli.2009.0182","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"28174363","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Selective protection of nuclease-sensitive sites in siRNA prolongs silencing effect.","authors":"Anton A Volkov,&nbsp;Natal'ya S Kruglova,&nbsp;Mariya I Meschaninova,&nbsp;Alya G Venyaminova,&nbsp;Marina A Zenkova,&nbsp;Valentin V Vlassov,&nbsp;Elena L Chernolovskaya","doi":"10.1089/oli.2008.0162","DOIUrl":"https://doi.org/10.1089/oli.2008.0162","url":null,"abstract":"<p><p>Small interfering RNAs (siRNAs) are considered as potent agents for specific gene silencing; however, nuclease sensitivity of siRNA limits their biomedical applications. Till date, no universal methodology has been developed to improve the nuclease resistance of siRNA, preserving low toxicity and high activity. In this study, we proposed an algorithm for the site-specific modification of siRNAs based on the mapping of their nuclease-sensitive sites in the presence of serum followed by the incorporation of 2'-O-methyl analogs of ribonucleotides at the identified positions of cleavage. We found that the protection of nuclease-sensitive sites considerably enhanced nuclease resistance of siRNA and only slightly reduced the efficiency of silencing. Modification of all nuclease-sensitive sites prolonged the duration of the silencing effect of the siRNA compared to nonmodified, partially modified, or randomly modified siRNA of the same sequence. This study showed that the targeted chemical modification of nuclease-sensitive sites could provide highly efficient siRNA-based therapeutics for the control of disease-related genes.</p>","PeriodicalId":19523,"journal":{"name":"Oligonucleotides","volume":"19 2","pages":"191-202"},"PeriodicalIF":0.0,"publicationDate":"2009-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/oli.2008.0162","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"28092016","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Stem-loop RT-PCR quantification of siRNAs in vitro and in vivo.","authors":"Angie Cheng,&nbsp;Mu Li,&nbsp;Yu Liang,&nbsp;Yu Wang,&nbsp;Linda Wong,&nbsp;Caifu Chen,&nbsp;Alexander V Vlassov,&nbsp;Susan Magdaleno","doi":"10.1089/oli.2008.0176","DOIUrl":"https://doi.org/10.1089/oli.2008.0176","url":null,"abstract":"<p><p>RNA interference (RNAi) is a mechanism in which the introduction of small interfering RNAs (siRNAs) into a diverse range of organisms and cell types causes degradation of the complementary mRNA. Applications of RNAi include gene function and pathway analysis, target identification and validation, and therapeutics. There is a need to develop reliable and easy-to-use assays to evaluate siRNA delivery efficiency and distribution, study pathways, and stability of siRNAs in cells (posttransfection) and in animals (postinjection). We have leveraged the Applied Biosystems TaqMan-based stem-loop RT-PCR technology, originally developed for quantification of endogenous microRNAs in cells, to fulfill these needs. The application protocols developed enable robust quantification of siRNA, including chemically modified siRNA molecules, in vitro and in vivo.</p>","PeriodicalId":19523,"journal":{"name":"Oligonucleotides","volume":"19 2","pages":"203-8"},"PeriodicalIF":0.0,"publicationDate":"2009-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/oli.2008.0176","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"28119735","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Naked siLNA-mediated gene silencing of lung bronchoepithelium EGFP expression after intravenous administration.","authors":"Sys Zoffmann Glud,&nbsp;Jesper B Bramsen,&nbsp;Frederik Dagnaes-Hansen,&nbsp;Jesper Wengel,&nbsp;Kenneth Alan Howard,&nbsp;Jens R Nyengaard,&nbsp;Jørgen Kjems","doi":"10.1089/oli.2008.0175","DOIUrl":"https://doi.org/10.1089/oli.2008.0175","url":null,"abstract":"<p><p>The use of systemic siRNA therapeutics for RNA interference-mediated silencing of disease genes is limited by serum instability and inadequate biodistribution. We have previously reported on the EGFP gene silencing effect of chitosan/siRNA nanoparticles in the bronchoepithelium of mice lungs following intranasal delivery and improved serum stability and reduced off-targeting effects in vitro by incorporation of locked nucleic acid (LNA). In this study, we examine the pulmonary gene silencing effect of siLNAs targeting enhanced-green-fluorescent-protein (EGFP) in lung bronchoepithelium upon intravenous delivery of naked siLNAs and upon intranasal delivery of either naked siLNA or chitosan/siLNA nanoparticles. We show that naked siLNA administered intravenously efficiently reduces the EGFP protein expression. A similar effect is obtained with intranasal delivery of chitosan nanoparticles containing siLNA whereas intranasally instilled naked siLNA did not cause a knockdown.</p>","PeriodicalId":19523,"journal":{"name":"Oligonucleotides","volume":"19 2","pages":"163-8"},"PeriodicalIF":0.0,"publicationDate":"2009-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/oli.2008.0175","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"28173713","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Inhibition of tumor growth and metastasis by non-small cell lung cancer cells transfected with cyclin D1-targeted siRNA.","authors":"Hu Huang,&nbsp;Yi-de Hu,&nbsp;Na Li,&nbsp;Yong Zhu","doi":"10.1089/oli.2008.0174","DOIUrl":"10.1089/oli.2008.0174","url":null,"abstract":"<p><p>To observe whether cyclin D1 siRNA-mediated inhibition of cyclin D1 represents a promising antigrowth and antimetastatic strategy for cancer gene therapy, particularly for non-small cell lung cancers. To stably transfect the A549 cell line with a cyclin D1-targeted siRNA to downregulate cyclin D1 expression and observe the effects on protein expression, and tumor growth in vitro and in vivo. Expression of cyclin D1-targeted siRNA resulted in a decrease in cyclin D1, MMP-2, RhoA, and Rac1 protein levels, as detected by Western blot and immunofluorescence studies. Transfected cells also exhibited a marked decrease in the rate of cell growth, and decreased invasive capacity, compared to cells transduced with a scrambled siRNA plasmid and untransduced A549 cells. siRNA-mediated inhibition of cyclin D1 expression represents a promising antigrowth and antimetastatic strategy for cancer gene therapy, particularly for non-small cell lung cancers. It is the reason for inhibiting tumor growth so that cyclin D1 siRNA can inhibit the cell cycle progression. In addition, the mechanism of inhibiting tumor metastasis was related to the decrease in the expression of MMP-2, RhoA, and Rac1 after cyclin D1 was decreased by cyclin D1 siRNA.</p>","PeriodicalId":19523,"journal":{"name":"Oligonucleotides","volume":"19 2","pages":"151-62"},"PeriodicalIF":0.0,"publicationDate":"2009-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/oli.2008.0174","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"28100733","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Analysis of putative miRNA binding sites and mRNA 3' ends as targets for siRNA-mediated gene knockdown.","authors":"Tobias Bergauer,&nbsp;Ute Krueger,&nbsp;Eric Lader,&nbsp;Sabrina Pilk,&nbsp;Irene Wolter,&nbsp;Wolfgang Bielke","doi":"10.1089/oli.2008.0154","DOIUrl":"https://doi.org/10.1089/oli.2008.0154","url":null,"abstract":"<p><p>Tremendous efforts have been made to develop short-interfering RNA (siRNA) design algorithms that generate highly functional siRNAs for gene knockdown. Nevertheless, \"difficult-to-silence\" target messenger RNAs (mRNAs) still exist for which no functionally validated siRNAs are available. MicroRNA (miRNA) sites in the mRNA 3'UTR, which interact with miRNA-loaded RNA-induced silencing complex (miRISC) for posttranscriptional gene regulation, provide alternative potentially accessible sites for siRNA. To investigate this, we designed siRNAs directed against single putative miRNA sites (misiRNAs) as predicted by miRNA target databases as well as siRNAs against other regions within the 3'UTR of \"difficult-to-silence\" targets in this proof-of-principle study. Although their design was not fully optimized, the misiRNAs generally caused higher knockdown than previously designed siRNAs for these targets. In general, knockdown by misiRNAs targeting the miRNA seed region was specific for the target mRNA, and misiRNAs targeting 1 nt upstream of miRNA seed region were similarly potent. We also systematically screened the 3'UTR of two mRNA targets using siRNA-tiling experiments. 5' and 3' regions of the p21-activated kinase 6 (PAK6) 3'UTR were found accessible, whereas the middle portion was largely inaccessible for siRNA knockdown. In ribosomal protein S6 kinase (RPS6KB1) 3'UTR, however, only the 5' region was accessible for siRNA knockdown. Detailed analysis of 10 further \"difficult-to-silence\" targets revealed that siRNA accessibility at the mRNA 3' end is not a general phenomenon, at least in \"difficult-to-silence\" targets, as we could not detect significant knockdown by siRNAs directed against this region.</p>","PeriodicalId":19523,"journal":{"name":"Oligonucleotides","volume":"19 1","pages":"41-52"},"PeriodicalIF":0.0,"publicationDate":"2009-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/oli.2008.0154","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27968550","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Benzimidazoles: a minor groove-binding ligand-induced stabilization of triple helix.","authors":"Akash K Jain,&nbsp;Sharad K Gupta,&nbsp;Urmila Tawar,&nbsp;Sneh K Dogra,&nbsp;Vibha Tandon","doi":"10.1089/oli.2008.0169","DOIUrl":"https://doi.org/10.1089/oli.2008.0169","url":null,"abstract":"<p><p>Nonintercalating minor groove-binding ligands netropsin, Hoechst 33258, and DAPI are reported to destabilize the triplex. Ligands with different substitutions on the phenyl ring of bis- and terbenzimidazoles were evaluated for their effect on the stability of C+.GC triplex and Hoogsteen duplex. We found that newly synthesized benzimidazoles stabilize the triplex as shown by fluorescence and melting studies. Modeling studies showed that these molecules bind in the Watson-Crick minor groove of triplex, which can exert a profound impact on the properties of the host triplex. Circular dichroism-binding studies indicate 5.77 base triplets/ligand as an apparent binding site for bis- and 8.66 for terbenzimidazoles. The stabilization of triplex can be attributed to the protonation of nitrogens and amines of benzimidazoles at pH 5.2 that compensate the negative charge of phosphate backbone to reduce the repulsion between the strands resulting in the stabilization.</p>","PeriodicalId":19523,"journal":{"name":"Oligonucleotides","volume":"19 1","pages":"53-62"},"PeriodicalIF":0.0,"publicationDate":"2009-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/oli.2008.0169","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27997385","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}